Use of gp130 activators in diabetic neuropathy

ABSTRACT

The invention relates to the use a substance signaling through gp130 for the manufacture of a medicament for the treatment and/or prevention of diabetic neuropathy. The use of IL-6 is preferred.

FIELD OF THE INVENTION

The present invention is in the field of diabetes mellitus andperipheral nervous system disorders. In particular, it relates to theuse of substances signaling through gp130 for the manufacture of amedicament for the treatment and/or prevention of diabetic neuropathy.IL-6, or an IL-6R/IL-6 chimera are preferably used in this specificmedical indication.

BACKGROUND OF THE INVENTION

Diabetes mellitus is a disorder of carbohydrate metabolism, i.e. asyndrome characterized by hyperglycemia resulting from absolute orrelative impairment in insulin secretion and/or insulin action.

Classification of Diabetes mellitus is based on the one adopted by theNational Diabetes Data Group and WHO. Previously, it was based on age atonset, duration, and complications of the disease. Gestational diabetesmellitus is carbohydrate intolerance of variable severity with onset orfirst recognition during the current pregnancy. Patients with type Idiabetes mellitus (DM), also known as insulin-dependent DM (IDDM) orjuvenile-onset diabetes, may develop diabetic ketoacidosis (DKA).Patents with type II DM, also known as non-insulin-dependent DM (NIDDM),may develop nonketotic hyperglycemic-hyperosmolar coma (NKHHC). Commonlate microvascular complications include retinopathy, nephropathy, andperipheral and autonomic neuropathies. Macrovascular complicationsinclude atherosclerotic coronary and peripheral arterial disease.

Type I diabetes mellitus: Although it may occur at any age, type Idiabetes mellitus most commonly develops in childhood or adolescence andis the predominant type of DM diagnosed before age 30. This type ofdiabetes accounts for 10 to 15% of all cases of DM and is characterizedclinically by hyperglycemia and a propensity to diabetic ketoacidosis.The pancreas produces little or no insulin.

About 80% of patients with type I DM have specific HLA phenotypesassociated with detectable serum islet cell cytoplasmic antibodies andislet cell surface antibodies (antibodies to glutamic acid decarboxylaseand to insulin are found in a similar proportion of cases).

In these patients, type I DM results from a genetically susceptible,immune-mediated, selective destruction of >90% of theirinsulin-secreting cells. Their pancreatic islets exhibit insulitis,which is characterized by an infiltration of T lymphocytes accompaniedby macrophages and B lymphocytes and by the loss of most of thebeta-cells, without involvement of the glucagon-secreting alpha-cells.The antibodies present at diagnosis usually become undetectable after afew years. They may be primarily a response to beta-cell destruction,but some are cytotoxic for beta-cells and may contribute to their loss.The clinical onset of type I DM may occur in some patients years afterthe insidious onset of the underlying autoimmune process. Screening forthese antibodies is included in numerous ongoing preventive studies.

Type II diabetes mellitus: Type II DM is usually the type of diabetesdiagnosed in patients >30 years, but it also occurs in children andadolescents. It is characterized clinically by hyperglycemia and insulinresistance. Diabetic ketoacidosis is rare. Although most patients aretreated with diet, exercise, and oral drugs, some patientsintermittently or persistently require insulin to control symptomatichyperglycemia and prevent nonketotic hyperglycemic-hyperosmolar coma.The concordance rate for type II DM in monozygotic twins is >90%. TypeII DM is commonly associated with obesity, especially of the upper body(visceral/abdominal), and often present after a period of weight gain.Impaired glucose tolerance associated with aging is closely correlatedwith the typical weight gain. Type II DM patients withvisceral/abdominal obesity may have normal glucose levels after losingweight.

Type II DM is a heterogeneous group of disorders in which hyperglycemiaresults from both an impaired insulin secretory response to glucose anddecreased insulin effectiveness in stimulating glucose uptake byskeletal muscle and in restraining hepatic glucose production (insulinresistance). However, insulin resistance is common, and most patientswith insulin resistance will not develop diabetes, because the bodycompensates by adequately increasing insulin secretion. Insulinresistance in the common variety of type II DM is not the result ofgenetic alterations in the insulin receptor or the glucose transporter.However, genetically determined post-receptor intracellular defectslikely play a role. The resulting hyperinsulinemia may lead to othercommon conditions, such as obesity (abdominal), hypertension,hyperlipidemia, and coronary artery disease (the syndrome of insulinresistance).

Genetic factors appear to be the major determinants for the developmentof type II DM, yet no association between type II DM and specific HLAphenotypes or islet cell cytoplasmic antibodies has been demonstrated.An exception is a subset of non-obese adults with detectable islet cellcytoplasmic antibodies who carry one of the HLA phenotypes and who mayeventually develop type I DM.

Before diabetes develops, patients generally lose the early insulinsecretory response to glucose and may secrete relatively large amountsof proinsulin. In established diabetes, although fasting plasma insulinlevels may be normal or even increased in type II DM patients,glucose-stimulated insulin secretion is dearly decreased. The decreasedinsulin levels reduce insulin-mediated glucose uptake and fail torestrain hepatic glucose production.

Hyperglycemia may not only be a consequence but also a cause of furtherimpairment in glucose tolerance in the diabetic patient (glucosetoxicity) because hyperglycemia decreases insulin sensitivity andincreases hepatic glucose production. Once a patient's metabolic controlimproves the insulin or hypoglycemic drug dose is usually lowered.

Some cases of type II DM occur in young, non-obese adolescents(maturity-onset diabetes of the young [MODY]) with an autosomal dominantinheritance. Many families with MODY have a mutation in the glucokinasegene. Impairments in insulin secretion and in hepatic glucose regulationhave been demonstrated in these patients.

Insulinopathies are rare cases of DM, with the clinical characteristicsof type II DM, result from the heterozygous inheritance of a defectivegene, leading to secretion of insulin that does not bind normally to theinsulin receptor. These patients have greatly elevated plasmaimmunoreactive insulin levels associated with normal plasma glucoseresponses to exogenous insulin.

Diabetes may also be attributed to pancreatic disease: Chronicpancreatitis, particularly in alcoholics, is frequently associated withdiabetes. Such patients lose both insulin-secreting andglucagon-secreting islets. Therefore, they may be mildly hyperglycemicand sensitive to low doses of insulin. Given the lack of effectivecounterregulation (exogenous insulin that is unopposed by glucagon),they frequently suffer from rapid onset of hypoglycemia. In Asia,Africa, and the Caribbean, DM is commonly observed in young, severelymalnourished patients with severe protein deficiency and pancreaticdisease; these patients are not prone to diabetic ketoacidosis but mayrequire insulin.

Diagnosis of diabetes mellitus: In asymptomatic patients, DM isestablished when the diagnostic criterion for fasting hyperglycemia ismet: a plasma (or serum) glucose level of >=140 mg/dl (>=7.77 mmol/l)after an overnight fast on two occasions in an adult or child.

An oral glucose tolerance test may be helpful in diagnosing type II DMin patients whose fasting glucose is between 115 and 140 mg/dl (6.38 and7.77 mmol/L) and in those with a clinical condition that might berelated to undiagnosed DM (e.g. polyneuropathy, retinopathy).

Treatment of diabetes mellitus: Hyperglycemia is responsible for most ofthe long-term microvascular complications of diabetes. It demonstrated alinear relationship between the levels of Hb A_(1c) (see below) and therate at which complications developed. Other studies have suggested thatHb A_(1c)<8% is a threshold below which most complications can beprevented. Thus, therapy for type I DM should try to intensify metaboliccontrol to lower Hb A_(1c) while avoiding hypoglycemic episodes.However, treatment must be individualized and should be modified whencircumstances make any risk of hypoglycemia unacceptable (e.g. inpatients with a short life expectancy and in those with cerebrovascularor cardiac disease) or when the patient's risk of hypoglycemia isincreased (e.g. in patients who are unreliable or who have autonomicneuropathy).

Diet to achieve weight reduction is most important in overweightpatients with type II DM. If improvement in hyperglycemia is notachieved by diet, trial with an oral drug should be started.

The patient should be regularly assessed for symptoms or signs ofcomplications, including a check of feet and pulses and sensation in thefeet and legs, and a urine test for albumin. Periodic laboratoryevaluation includes lipid profile, BUN (blood urea nitrogen) and serumcreatinine levels, ECG, and an annual complete ophthalmologicevaluation.

Hypercholesterolemia or hypertension increases the risks for specificlate complications and requires special attention and appropriatetreatment. Although beta-adrenergic receptor blocking agents β-blockers,such as propranolol) can be used safely in most diabetics, they can maskthe β-adrenergic symptoms of insulin-induced hypoglycemia and can impairthe normal counterregulatory response. Thus, ACE inhibitors and calciumantagonists are often the drugs of choice.

Plasma glucose monitoring should be carried out by all patients, andinsulin-treated patients should be taught to adjust their insulin dosesaccordingly. Glucose levels can be tested with easy-to-use homeanalyzers using a drop of fingertip blood. A spring-powered lancet isrecommended to obtain the fingertip blood sample. The frequency oftesting is determined individually. Insulin-treated diabetic patientsideally should test their plasma glucose daily before meals, 1 to 2hours after meals, and at bedtime.

Most physicians periodically determine glycosylated hemoglobin (HbA_(1c)) to estimate plasma glucose control during the preceding 1 to 3months. Hb A_(1c) is the stable product of non-enzymatic glycosylationof Hb by plasma glucose and is formed at rates that increase withincreasing plasma glucose levels. In most laboratories, the normal HbA_(1c) level is about 6%; in poorly controlled diabetics, the levelranges from 9 to 12%. Hb A_(1c) is not a specific test for diagnosingdiabetes; however, elevated Hb A_(1c) often indicates existing diabetes.

Another test determines the fructosamine level. Fructosamine is formedby a chemical reaction of glucose with plasma protein and reflectsglucose control in the previous 1 to 3 weeks. Therefore, this assay mayshow a change in control before Hb A_(1c) and is often helpful whenintensive treatment is applied and in short-term clinical trials.

As regards insulin treatment, human insulin is often preferred ininitiating insulin treatment because it is less antigenic thananimal-derived varieties. However, detectable insulin antibody levels,usually very low, develop in most insulin-treated patients, includingthose receiving human insulin preparations.

Insulin is routinely provided in preparations containing 100 U/ml (U-100insulin) and is injected subcutaneously with disposable insulinsyringes. The ½-ml syringes are generally preferred by patients whoroutinely inject doses of <=50 U, because they can be read more easilyand facilitate the accurate measurement of smaller doses. Amultiple-dose insulin injection device (NovolinPen), commonly referredto as an insulin pen, is designed to use a cartridge containing severaldays' dosage. Insulin should be refrigerated but never frozen; however,most insulin preparations are stable at room temperature for months,which facilitates their use at work and when traveling.

Diabetes may be associated with other endocrine diseases. Type II DM canbe secondary to Cushing's syndrome, acromegaly, pheochromocytoma,glucagonoma, primary aldosteronism, or somatostatinoma. Most of thesedisorders are associated with peripheral or hepatic insulin resistance.Many patients will become diabetic once insulin secretion is alsodecreased. The prevalence of type I DM is increased in patients withcertain autoimmune endocrine diseases, e.g. Graves' disease, Hashimoto'sthyroiditis, and idiopathic Addison's disease.

Diabetes may also be induced by beta-cell toxins. Streptozotocin forinstance can induce experimental diabetes in rats but rarely causesdiabetes in humans.

Late complications of diabetes occur after several years of poorlycontrolled hyperglycemia. Glucose levels are increased in all cellsexcept where there is insulin-mediated glucose uptake (mainly muscle),resulting in an increase in glycolysation and in the activity of othermetabolic pathways, which may be caused by complications. Mostmicrovascular complications can be delayed, prevented, or even reversedby tight glycemic control, i.e. achieving near-normal fasting andpostprandial glucose levels, reflected by near-normal glycosylatedhemoglobin (Hb A_(1c)). Macrovascular disease such as atherosclerosismay lead to symptomatic coronary artery disease, claudication, skinbreakdown, and infections. Although hyperglycemia may accelerateatherosclerosis, many years of hyperinsulinemia preceding the onset ofdiabetes (with insulin resistance) may play a major initiating role.Amputation of a lower limb for severe peripheral vascular disease,intermittent claudication, and gangrene remains common. Backgroundretinopathy (the initial retinal changes seen on ophthalmoscopicexamination or in retinal photographs) does not significantly altervision, but it can progress to macular edema or proliferativeretinopathy with retinal detachment or hemorrhage, which can causeblindness. About 85% of all diabetics eventually develop some degree ofretinopathy. Diabetic nephropathy is usually asymptomatic untilend-stage renal disease develops, but it can cause the nephroticsyndrome.

Diabetic neuropathy is a further complication of diabetes, but it isalso common in connection with other diseases.

Multiple mononeuropathy is usually secondary to collagen vasculardisorders (e.g. polyarteritis nodosa, systemic lupus erythematosus(SLE), Sjögren's syndrome, rheumatoid arthritis (RA)), sarcoidosis,metabolic diseases (e.g. diabetes, amyloidosis), or infectious diseases(e.g. Lyme disease, HIV infection). Microorganisms may cause multiplemononeuropathy by direct invasion of the nerve (e.g. in leprosy).

Polyneuropathy due to acute febrile diseases may result from a toxin(e.g. in diphtheria) or an autoimmune reaction (e.g. in Guillain-Barrésyndrome); the polyneuropathy that sometimes follows immunizations isprobably also autoimmune.

Toxic agents generally cause polyneuropathy but sometimesmononeuropathy. They include emetine, hexobarbital, barbital,chlorobutanol, sulfonamides, phenytoin, nitrofurantoin, the vincaalkaloids, heavy metals, carbon monoxide, triorthocresyl phosphate,orthodinitrophenol, many solvents, other industrial poisons, and certainAIDS drugs (e.g. zalcitabine, didanosine).

Nutritional deficiencies and metabolic disorders may result inpolyneuropathy. B vitamin deficiency is often the cause (e.g. inalcoholism, beriberi, pernicious anemia, isoniazid-induced pyridoxinedeficiency, malabsorption syndromes, and hyperemesis gravidarum).Polyneuropathy also occurs in hypothyroidism, porphyria, sarcoidosis,amyloidosis, and uremia.

Malignancy may cause polyneuropathy via monoclonal gammopathy (multiplemyeloma, lymphoma), amyloid invasion, or nutritional deficiencies or asa paraneoplastic syndrome.

Polyneuropathy due to metabolic disorders, such as diabetes mellitus orrenal failure, develops slowly, often over months or years. Itfrequently begins with sensory abnormalities in the lower extremitiesthat are often more severe distally than proximally. Peripheraltingling, numbness, burning pain, or deficiencies in jointproprioception and vibratory sensation are often prominent. Pain isoften worse at night and may be aggravated by touching the affected areaor by temperature changes. In severe cases, there are objective signs ofsensory loss, typically with stocking-and-glove distribution. Achillesand other deep tendon reflexes are diminished or absent. Painless ulcerson the digits or Charcot's joints may develop when sensory loss isprofound. Sensory or proprioceptive deficits may lead to gaitabnormalities. Motor involvement results in distal muscle weakness andatrophy. The autonomic nervous system may be additionally or selectivelyinvolved, leading to nocturnal diarrhea, urinary and fecal incontinence,impotence, or postural hypotension. Vasomotor symptoms vary. The skinmay be paler and drier than normal, sometimes with dusky discoloration;sweating may be excessive. Trophic changes (smooth and shiny skin,pitted or ridged nails, osteoporosis) are common in severe, prolongedcases.

Treatment of the systemic disorder (e.g. diabetes mellitus, renalfailure, multiple myeloma, tumor) may halt progression and improvesymptoms, but recovery is slow. Entrapment neuropathies may requirecorticosteroid injections or surgical decompression. Physical therapyand splints reduce the likelihood or severity of contractures.

Diabetes mellitus can cause sensorimotor distal polyneuropathy (mostcommon), multiple mononeuropathy, and focal mononeuropathy (e.g. of theoculomotor or abducens cranial nerves). Polyneuropathy commonly occursas a distal, symmetric, predominantly sensory polyneuropathy that causessensory deficits, which begin with and are usually marked by astocking-glove distribution.

Generally, peripheral neuropathy is defined as a syndrome of sensoryloss, muscle weakness and atrophy, decreased deep tendon reflexes, andvasomotor symptoms, alone or in any combination. The disease may affecta single nerve (mononeuropathy), two or more nerves in separate areas(multiple mononeuropathy), or many nerves simultaneously(polyneuropathy). The axon may be primarily affected (such as indiabetes mellitus, Lyme disease, or uremia or with toxic agents) or themyelin sheath or Schwann cell (such as in acute or chronic inflammatorypolyneuropathy, leukodystrophies, or Guillain-Barré syndrome). Damage tosmall unmyelinated and myelinated fibers results primarily in loss oftemperature and pain sensation; damage to large myelinated fibersresults in motor or proprioceptive defects. Some neuropathies (e.g. dueto lead toxicity, dapsone use, tick bite, porphyria, or Guillain-Barrésyndrome) primarily affect motor fibers; others (e.g. due to dorsal rootganglionitis of cancer, leprosy, AIDS, diabetes mellitus, or chronicpyridoxine intoxication) primarily affect the dorsal root ganglia orsensory fibers, producing sensory symptoms. Occasionally, cranial nervesare also involved (e.g. in Guillain-Barré syndrome, Lyme disease,diabetes mellitus, and diphtheria).

Trauma is the most common cause of a localized injury to a single nerve.Violent muscular activity or forcible overextension of a joint mayproduce a focal neuropathy, as may repeated small traumas (e.g. tightgripping of small tools, excessive vibration from air hammers). Pressureor entrapment paralysis usually affects superficial nerves (ulnar,radial, peroneal) at bony prominences (e.g. during sound sleep or duringanesthesia in thin or cachectic persons and often in alcoholics) or atnarrow canals (e.g. in carpal tunnel syndrome). Pressure paralysis mayalso result from tumors, bony hyperostosis, casts, crutches, orprolonged cramped postures (e.g. in gardening). Hemorrhage into a nerveand exposure to cold or radiation may also cause neuropathy.Mononeuropathy may further result from direct tumor invasion.

Diabetic polyneuropathy may cause numbness, tingling, and paresthesiasin the extremities and, less often, debilitating, severe, deep-seatedpain and hyperesthesias. Ankle jerks are usually decreased or absent.Other causes of polyneuropathy must be excluded. Acute, painfulmononeuropathies affecting the 3rd, 4th, or 6th cranial nerve as well asother nerves, such as the femoral, may spontaneously improve over weeksto months, occur more frequently in older diabetics, and are attributedto nerve infarctions. Autonomic neuropathy occurs primarily in diabeticswith polyneuropathy and can cause postural hypotension, disorderedsweating, impotence and retrograde ejaculation in men, impaired bladderfunction, delayed gastric emptying (sometimes with dumping syndrome),esophageal dysfunction, constipation or diarrhea, and nocturnaldiarrhea. A decrease in heart rate response to the Valsalva maneuver oron standing and unchanged heart rate variation during deep breathing areevidence of autonomic neuropathy in diabetics.

Diabetic polyneuropathy is the major cause for foot ulcers and jointproblems, which are important causes of morbidity in diabetes mellitus.In diabetic polyneuropathy, the sensory denervation impairs theperception of trauma from such common causes as ill-fitting shoes orpebbles. Alterations in proprioception lead to an abnormal pattern ofweight bearing and sometimes to the development of Charcot's joints.

Patients with infected foot ulcers frequently feel no pain because ofneuropathy and have no systemic symptoms until late in a neglectedcourse. Deep ulcers and particularly ulcers associated with anydetectable cellulitis require immediate hospitalization, since systemictoxicity and permanent disability may develop. Early surgicaldebridement is an essential part of management, but amputation issometimes necessary.

Interleukin-6 (IL-6) is a multifunctional cytokine produced and secretedby several different cell types. This pleiotropic cytokine plays acentral role in cell defense mechanisms including the immune response,acute phase response and hematopoiesis. IL-6 is a 20 to 26 kDaglycoprotein having 185 amino acids that has been cloned previously (Mayet al, (1986); Zilberstein et al, (1986); Hirano et al, (1986)). IL-6has previously been referred to as B cell stimulatory factor 2 (BSF-2),interferon-beta 2 and hepatocyte stimulatory factor. IL-6 is secreted bya number of different tissues including the liver, spleen, and bonemarrow and by a variety of cell types including monocytes, fibroblasts,endothelial, B- and T-cells. IL-6 is activated at the transcriptionallevel by a variety of signals including viruses, double stranded RNA,bacteria and bacterial lipopolysaccarides, and inflammatory cytokinessuch as IL-1 and TNF.

IL-6 has been implicated in the pathogenesis of human inflammatory CNSdiseases. Increased plasma and cerebrospinal fluid levels of IL-6 havebeen demonstrated in patients with multiple sclerosis (Frei et al.,(1991)), for instance.

Recent experiments on the effects of IL-6 on cells of the central andperipheral nervous system indicate that IL-6 may have protective effectson neuronal cells as well as some impact on inflammatoryneurodegenerative processes (Gadient and Otten, 1997, Mendel et al,1998). IL-6 was found to prevent glutamate-induced cell death inhippocampal (Yamada et al., 1994) as well as in striatal (Toulmond etal., 1992) neurons. In transgenic mice expressing high levels of bothhuman IL-6 and human soluble IL-6R (sIL-6-R), an accelerated nerveregeneration was observed following injury of the hypoglossal nerve asshown by retrograde labeling of the hypoglossal nuclei in the brain(Hirota et al, 1996). Furthermore, there has been some evidence thatIL-6 is implied in a neurological disease, the demyelinating disorderMultiple Sclerosis (MS) (Mendel et al., 1998). Mice lacking the IL-6gene were resistant to the experimental induction of the disease. On theother hand, there have been reports indicating that IL-6 has a negativeeffect on neuronal survival during early post-traumatic phase afternerve injury (Fisher et al., 2001)

The biological activities of IL-6 are mediated by a membrane receptorsystem comprising two different proteins one named IL-6 receptor or gp80and the other gp130 (reviewed by Hirano et al, 1994). gp130 is atransmembrane glycoprotein with a length of 918 amino acids, includingan intracellular domain of 277 amino acids, is a subunit constituent ofseveral cytokine receptors, including those for IL-6, IL-11, LIF,Oncostatin M, CNTF (ciliary neurotrophic factor), CT-1. IL-6 being theprototype of the cytokines acting through gp130, this cytokine family isalso called “IL-6 type cytokines”.

gp130 participates in the formation of high-affinity receptors for thesecytokines by binding to low affinity receptor chains. Accordingly, gp130has been called also an “affinity converter”. Ligand binding to acytokine receptor leads to the dimerization of gp130 (shown for the IL6receptor) or heterodimerization (shown for LIF, Oncostatin M, and CNTFreceptors) with a gp130-related protein known as the LIFRbeta subunit.Binding of the respective ligands is associated with theactivation/association of a family of tyrosine kinases known as Januskinases (JAKs), as the first step of intracellular signal transduction.Intracellular signaling processes include tyrosine phosphorylation andactivation factors called STATs (signal transducer and activator oftranscription).

The human gp130 gene product appears to be homologous to two distinctchromosomal loci on chromosomes 5 and 17. The presence of two distinctgp130 gene sequences is restricted to primates and is not found in othervertebrates.

It has been shown that the signaling activities of IL-6, IL-11, CNTF,Oncostatin M and LIF can be blocked specifically by different monoclonalantibodies directed against gp130. In addition to this, monoclonalantibodies, which directly activate gp130 independently of the presenceof cytokines or their receptors have been found.

Other monoclonal antibodies directed against gp130 have been shown toinhibit IL-6-mediated functions. Soluble forms of gp130 (sgp130) withmolecular masses of 90 and 110 Kda have been found in human serum. Theycan inhibit biological functions of those cytokines utilizing receptorsystems with gp130 as a component.

Soluble forms of IL-6R gp80 (sIL-6R), corresponding to the extracellulardomain of gp80, are natural products of the human body found asglycoproteins in blood and in urine (Novick et al, 1990, 1992). Anexceptional property of sIL-6R molecules is that they act as potentagonists of IL-6 on many cell types including human cells (Taga et al,1989; Novick et al, 1992). Even without the intracytoplasmic domain ofgp80, sIL-6R is still capable of triggering the dimerization of gp130 inresponse to IL-6, which in turn mediates the subsequent IL-6-specificsignal transduction and biological effects (Murakami et al, 1993).sIL-6R has two types of interaction with gp130 both of which areessential for the IL-6 specific biological activities (Halimi et al.,1995), and the active IL-6 receptor complex was proposed to be ahexameric structure formed by two gp130 chains, two IL-6R and two IL-6ligands (Ward et al., 1994; Paonessa et al, 1995).

Chimeric molecules linking the soluble IL-6 receptor and IL-6 togetherhave been described (Chebath et al., 1997, Fischer et al., 1997, WO99/02552 and WO 97/32891). They have been designated IL-6R/IL-6 chimeraand Hyper-IL-6, respectively, and will be called IL-6R/IL-6 in thefollowing. The IL-6R/IL-6 chimera were generated by fusing the entirecoding regions of the cDNAs encoding the soluble IL-6 receptor (sIL-6R)and IL-6 (Fischer et al., 1997; Chebath et al., 1997). RecombinantIL-6R/IL-6 chimera was produced in CHO cells (Chebath et al, 1997,WO99/02552). IL-6R/IL-6 chimera binds with a higher efficiency to thegp130 chain in vitro than does the mixture of IL-6 with sIL-6R (Kolletet al, 1999).

SUMMARY OF THE INVENTION

In accordance with the present invention it has been found that theadministration of substances signaling through gp130 resulted in asignificant beneficial effect in an established animal model of diabeticneuropathy. Exemplary substances tested were IL-6 and an IL-6R/IL-6chimera. Both substances showed a statistically significant beneficialeffect in diabetic neuropathy, as indicated by the improvement ofseveral parameters relating to nerve vitality.

The invention therefore relates to the use of a substance signalingthrough gp130 for the preparation of a medicament for treatment and/orprevention of diabetic neuropathy.

The use of cells expressing substances signaling through gp130 for themanufacture of a medicament for the treatment and/or prevention ofdiabetic neuropathy is a further object of the present invention.Furthermore, in accordance with the present invention, vectorscomprising the coding sequences for substances signaling through gp130are used for the manufacture of a medicament for the treatment and/orprevention of diabetic neuropathy

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the development of body weight in experimental animals.

FIG. 2 shows the extent of glycemia after day 10 (A) and day 40 (B) ofdiabetes induction in the experimental series of animals receivingintraperitoneal administration.

FIG. 3 shows the time taken by animals receiving intraperitonealadministration to flick their tail placed on a heat source in seconds.

FIG. 4 shows the compound muscle action potential (CMAP) of the animalsreceiving intraperitoneal administration expressed in latency persecond.

FIG. 5 shows the sensory nerve conduction velocity (SNVC) in m/sec inthe experimental animals receiving intraperitoneal administration.

FIG. 6 shows the axon diameter in micrometers in the experimentalanimals receiving intraperitoneal administration.

FIG. 7 shows the fiber diameter in micrometers in the experimentalanimals receiving intraperitoneal administration.

FIG. 8 shows the myelin thickness in micrometers in the experimentalanimals receiving intraperitoneal administration.

FIG. 9 shows the number of myelinated fibers per field in theexperimental animals receiving intraperitoneal administration.

FIG. 10 is a schematic drawing illustrating the IL-6R/IL-6 chimerastructure.

FIG. 11 shows the development of body weight in experimental animals ofgroup A of animals receiving subcutaneous administration.

FIG. 12 shows the extent of glycemia after day 10 and day 41 of diabetesinduction in experimental animals of group A of animals receivingsubcutaneous administration.

FIG. 13 shows the time to flick their tail placed on a heat source inseconds by experimental animals of group A of animals receivingsubcutaneous administration.

FIG. 14 shows the number of crossed squares (A) and rearings (B) inexperimental animals of group A of animals receiving subcutaneousadministration.

FIG. 15 shows the compound muscle action potential (CMAP) expressed inlatency per second in experimental animals of group A of animalsreceiving subcutaneous administration.

FIG. 16 shows the sensory nerve conduction velocity (SNVC) in m/secexperimental animals of group A of animals receiving subcutaneousadministration.

FIG. 17 shows the fiber diameter in micrometers in experimental animalsof group A of animals receiving subcutaneous administration.

FIG. 18 shows the axon diameter in micrometers in experimental animalsof group A of animals receiving subcutaneous administration.

FIG. 19 shows the myelin thickness in micrometers in experimentalanimals of group A of animals receiving subcutaneous administration.

FIG. 20 shows the percentage of degenerate fibers in experimentalanimals of group A of animals receiving subcutaneous administration.

FIG. 21 shows the percentage of myelinated fibers in experimentalanimals of group A of animals receiving subcutaneous administration.

FIG. 22 shows the development of body weight in experimental animals ofgroup B of animals receiving subcutaneous administration.

FIG. 23 shows the extent of glycemia after day 10 and day 40 of diabetesinduction in experimental animals of group B of animals receivingsubcutaneous administration.

FIG. 24 shows the time to flick their tail placed on a heat source inseconds by experimental animals of group B of animals receivingsubcutaneous administration.

FIG. 25 shows the number of crossed squares (A) and rearings (B) inexperimental animals of group B of animals receiving subcutaneousadministration.

FIG. 26 shows the compound muscle action potential (CMAP) expressed inlatency per second in experimental animals of group B of animalsreceiving subcutaneous administration.

FIG. 27 shows the sensory nerve conduction velocity (SNVC) in m/secexperimental animals of group B of animals receiving subcutaneousadministration.

FIG. 28 shows the fiber diameter in micrometer in experimental animalsof group B of animals receiving subcutaneous administration.

FIG. 29 shows the axon diameter in micrometer in experimental animals ofgroup B of animals receiving subcutaneous administration.

FIG. 30 shows the myelin thickness in micrometers in experimentalanimals of group B of animals receiving subcutaneous administration.

FIG. 31 shows the percentage of degenerate fibers in experimentalanimals of group B of animals receiving subcutaneous administration.

FIG. 32 shows the percentage of myelinated fibers in experimentalanimals of group B of animals receiving subcutaneous administration.

FIG. 33 shows the development of body weight in experimental animals ofgroup C of animals receiving subcutaneous administration.

FIG. 34 shows the extent of glycemia after day 10 and day 40 of diabetesinduction in experimental animals of group C of animals receivingsubcutaneous administration.

FIG. 35 shows the time to flick their tail placed on a heat source inseconds by experimental animals of group C of animals receivingsubcutaneous administration.

FIG. 36 shows the number of crossed squares (A) and rearings (B) inexperimental animals of group C of animals receiving subcutaneousadministration.

FIG. 37 shows the compound muscle action potential (CMAP) expressed inlatency per second in experimental animals of group C of animalsreceiving subcutaneous administration.

FIG. 38 shows the sensory nerve conduction velocity (SNVC) in m/secexperimental animals of group C of animals receiving subcutaneousadministration.

FIG. 39 shows the fiber diameter in micrometers in experimental animalsof group C of animals receiving subcutaneous administration.

FIG. 40 shows the axon diameter in micrometers in experimental animalsof group C of animals receiving subcutaneous administration.

FIG. 41 shows the myelin thickness in micrometers in experimentalanimals of group C of animals receiving subcutaneous administration.

FIG. 42 shows the percentage of degenerate fibers in experimentalanimals of group C of animals receiving subcutaneous administration.

FIG. 43 shows the percentage of myelinated fibers in experimentalanimals of group C of animals receiving subcutaneous administration.

DETAILED DESCRIPTION OF THE INVENTION

The invention is based on the finding that that the administration ofsubstances signaling through gp130 resulted in a significantantinociceptive and nerve regenerating effect in an established animalmodel of diabetic neuropathy. Therefore, the invention relates to theuse of a substance, which initiates signaling through the humaninterleukin-6. (IL-6) receptor gp130 for the preparation of a medicamentfor treatment and/or prevention of diabetic neuropathy.

A “substance signaling through gp130” as used herein is any moleculeactivating the signaling cascade through gp130, i.e. any agonist,stimulator or activator of the gp130 portion of the IL-6 receptorcomplex. Stimulation may be direct, i.e. activation may be triggered bybinding directly to gp130. An example for such a direct activator isIL-6R/IL-6 chimera. Stimulation may also be indirect by binding toanother cell surface receptor, which forms a complex with gp130 therebyactivating it IL-6 is an example for such an indirect activator ofgp130. Further examples of substances signaling through gp130 includeIL-11, LIF, Oncostatin M (OSM), CNTF (ciliary neurotrophic factor), andcardiotrophin-1 (CT-1), which are the so-called “IL-6-type cytokines”.These cytokines trigger the JAK/STAT pathway, the first event of whichis the ligand-induced homo- or hetero-dimerization of signal-transducingreceptor subunits. All IL-6-type cytokines recruit gp130 to theirreceptor complexes. They either signal via gp130 alone or in combinationwith LIFR or OSMR, which are all able to activate Jaks and to recruitSTAT proteins. IL-6 induces gp130-homodimerization, whereas CNTF, LIF,and CT-1 signal via heterodimerization of gp130 and LIFR.

The terms “treating” and “preventing” as used herein should beunderstood as preventing, inhibiting, attenuating, ameliorating orreversing one or more symptoms or cause(s) of diabetic neuropathy, aswell as symptoms, diseases or complications accompanying diabeticneuropathy. When “treating” diabetic neuropathy, the substancesaccording to the invention are given after onset of the disease,“prevention” relates to administration of the substances before anysigns of disease can be noted in the patient. Preventive administrationis especially useful in high-risk patients, such as those patientshaving suffered from diabetes mellitus already for a prolonged period oftime.

The term “diabetic neuropathy” relates to any form of diabeticneuropathy, or to one or more symptom(s) or disorder(s) accompanying orcaused by diabetic neuropathy, or complications of diabetes affectingnerves as described in detail in the introduction above.

In a preferred embodiment of the invention, the diabetic neuropathy is apolyneuropathy. In diabetic polyneuropathy, many nerves aresimultaneously affected.

In a further preferred embodiment, the diabetic neuropathy is amononeuropathy. In focal mononeuropathy, the disease affects a singlenerve, such as the oculomotor or abducens cranial nerve. The disorder iscalled multiple mononeuropathy when two or more nerves are affected inseparate areas.

Preferably, the substance is:

-   a) IL-6;-   b) a fragment of a) which binds to gp80 and initiates signaling    through gp130;-   c) a variant of a) or b) which has at least 70% sequence identity    with a) or b) and which initiates signaling through gp130;-   d) a variant of a) or b) which is encoded by a DNA sequence which    hybridizes to the complement of the native DNA sequence encoding a)    or b) under moderately stringent conditions and which initiates    signaling through gp130; or-   e) a salt, fused protein or functional derivative of a), b), c)    or d) which initiates signaling through gp130.

The use of IL-6 itself is highly preferred according to the invention.IL-6 can be native IL-6, i.e. IL-6 isolated from a natural source, orrecombinantly produced IL-6. Recombinant IL-6 is particularly preferredaccording to the invention.

In a further preferred embodiment of the invention, the substance is

-   a) An IL-6R/IL-6 chimera;-   b) a fragment of a) which initiates signaling through gp130;-   c) a variant of a) or b) which has at least 70% sequence identity    with a) or b) and initiates signaling through gp130;-   d) a variant of a) or b) which is encoded by a DNA sequence which    hybridizes to the complement of the DNA sequence encoding a) or b)    under moderately stringent conditions and initiates signaling    through gp130; or-   e) a salt, fused protein or functional derivative of a), b), c)    or d) which initiates signaling through gp130.

An “IL-6R/IL-6 chimera” (also called “IL-6R/IL-6” or “IL-6 chimera”), asused herein, is a chimeric molecule comprising a soluble part of gp130fused to all or a biologically active fraction of interleukin-6. Themoieties of the chimeric protein can be fused directly to one another,or they can be linked by any suitable linker, such as a disulfide bridgeor a polypeptide linker. The linker may be a short linker peptide whichcan be as short as 1 to 3 amino acid residues in length or longer, forexample, 13 or 18 amino acid residues in length. Said linker may be atripeptide of the sequence E-F-M (Glu-Phe-Met), for example, or a13-amino acid linker sequence comprisingGlu-Phe-Gly-Ala-Gly-Leu-Val-Leu-Gly-Gly-Gln-Phe-Met introduced betweenthe amino acid sequence of the soluble IL-6 receptor gp130 and the IL-6sequence. Examples of IL-6R/IL-6 chimera are known in the art and havebeen described in detail e.g. in WO 99/02552 or WO 97/32891. An examplefor an IL-6R/IL-6 chimeric molecule which can be used according to theinvention is depicted schematically in FIG. 2.

As used herein the term “variant” refers to analogs of IL-6 or anIL-6R/IL-6 chimera, in which one or more of the amino acid residues ofthe naturally occurring components of IL-6R/IL-6 are replaced bydifferent amino acid residues, or are deleted, or one or more amino acidresidues are added to the original sequence of IL-6 or an IL-6R/IL-6,without changing considerably the activity of the resulting products ascompared to the original IL-6 or IL-6R/IL-6 chimera. These variants areprepared by known synthesis and/or by site-directed mutagenesistechniques, or any other known technique suitable therefor.

Variants in accordance with the present invention include proteinsencoded by a nucleic acid, such as DNA or RNA, which hybridizes to thecomplement of the DNA or RNA encoding IL-6 or an IL-6R/IL-6 undermoderately stringent or stringent conditions. The term “stringentconditions” refers to hybridization and subsequent washing conditions,which those of ordinary skill in the art conventionally refer to as“stringent”. See Ausubel et al., Current Protocols in Molecular Biology,supra, Interscience, N.Y., §§6.3 and 6.4 (1987, 1992), and Sambrook etal. (Sambrook, J. C., Fritsch, E. F., and Maniatis, T. (1989) MolecularCloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y.).

Without limitation, examples of stringent conditions include washingconditions 12-20° C. below the calculated Tm of the hybrid under studyin, e.g. 2×SSC and 0.5% SDS for 5 minutes, 2×SSC and 0.1% SDS for 15minutes; 0.1×SSC and 0.5% SDS at 37° C. for 30-60 minutes and then, a0.1×SSC and 0.5% SDS at 68° C. for 30-60 minutes. Those of ordinaryskill in this art understand that stringency conditions also depend onthe length of the DNA sequences, oligonucleotide probes (such as 10-40bases) or mixed oligonucleotide probes. If mixed probes are used, it ispreferable to use tetramethyl ammonium chloride (TMAC) instead of SSC,see Ausubel, supra. “Moderately stringent conditions”, refer to washingconditions at lower temperatures, lower salt or lower detergentconcentrations, such as in 0.2×SSC/0.1% SDS at 42° C. (Ausubel et al.,1989, supra)

Any such variant preferably has a sequence of amino acids sufficientlyduplicative of that of IL-6 or an IL-6R/IL-6, such as to havesubstantially similar, or even better, activity as compared to IL-6 orIL-6R/IL-6.

A characteristic activity of IL-6 is its capability of binding to thegp80 portion of the IL-6 receptor, and a characteristic activity ofIL-6R/IL-6 chimera is its capability of binding to gp130. An ELISA typeassay for measuring the binding of IL-6R/IL-6 chimera to gp130 has beendescribed in detail in example 7 on page 39 of WO 99/02552, which isfully incorporated by reference herein. The person skilled in the artwill appreciate that a similar ELISA type assay can be developed for thebinding of IL-6 to gp80. As long as the variant has substantial bindingactivity to its respective binding region of gp80 or of gp130, it can beconsidered to have substantially similar activity to IL-6 or IL-6R/IL-6chimera. Thus, it can be determined whether any given variant has atleast substantially the same activity as IL-6 or IL-6R/IL-6 by means ofroutine experimentation comprising subjecting such a varaint, e.g. to asimple sandwich binding assay to determine whether or not it binds to animmobilized gp80 or gp130, as described in example 7 of WO 99/02552.

In a preferred embodiment, any such variant has at least 40% identity orhomology with the sequence of mature IL-6 or the IL-6R/IL-6 chimericmolecule comprised in WO 99/02552. More preferably, it has at least 50%,at least 60%, at least 70%, at least 80% or, most preferably, at least90% identity or homology thereto.

Identity reflects a relationship between two or more polypeptidesequences or two or more nucleotide sequences, determined by comparingthe sequences. In general, identity refers to an exact nucleotide tonucleotide or amino acid to amino acid correspondence of the twonucleotides or two polypeptide sequences, respectively, over the lengthof the sequences being compared.

For sequences where there is not an exact correspondence, a “% identity”may be determined. In general, the two sequences to be compared arealigned to give a maximum correlation between the sequences. This mayinclude inserting “gaps” in either one or both sequences, to enhance thedegree of alignment. A % identity may be determined over the wholelength of each of the sequences being compared (so-called globalalignment), that is particularly suitable for sequences of the same orvery similar length, or over shorter, defined lengths (so-called localalignment), that is more suitable for sequences of unequal length.

Methods for comparing the identity and homology of two or more sequencesare well known in the art. Thus for instance, programs available in theWisconsin Sequence Analysis Package, version 9.1 (Devereux J et al.1984), for example the programs BESTFIT and GAP, may be used todetermine the % identity between two nucleotides and the % identity andthe % homology between two polypeptide sequences. BESTFIT uses the“local homology” algorithm of Smith and Waterman (1981) and finds thebest single region of similarity between two sequences. Other programsfor determining identity and/or similarity between sequences are alsoknown in the art, for instance the BLAST family of programs (Altschul SF et al, 1990, Altschul S F et al, 1997, accessible through the homepage of the NCBI at www.ncbi.nim.nih.gov) and FASTA (Pearson W R, 1990;Pearson 1988).

Variants of IL-6 or IL-6R/IL-6 chimera, which can be used in accordancewith the present invention, or nucleic acid coding therefor, include afinite set of substantially corresponding sequences as substitutionpeptides or nucleotides which can be routinely obtained by one ofordinary skill in the art, without undue experimentation, based on theteachings and guidance presented herein.

Preferred changes for variants in accordance with the present inventionare what are known as “conservative” substitutions. Conservative aminoacid substitutions of IL-6 or IL-6R/IL-6 chimera may include synonymousamino acids within a group which have sufficiently similarphysicochemical properties that substitution between members of thegroup will preserve the biological function of the molecule (Grantham,1974). It is clear that insertions and deletions of amino acids may alsobe made in the above-defined sequences without altering their function,particularly if the insertions or deletions only involve a few aminoadds, e.g. under thirty, and preferably under ten, and do not remove ordisplace amino acids which are critical to a functional conformation,e.g. cysteine residues. Proteins and variants thereof produced by suchdeletions and/or insertions come within the purview of the presentinvention.

Preferably, the synonymous amino acid groups are those defined inTable 1. More preferably, the synonymous amino add groups are thosedefined in Table 2; and most preferably the synonymous amino acid groupsare those defined in Table 3. TABLE 1 Preferred Groups of SynonymousAmino Acids Amino Acid Synonymous Group Ser Ser, Thr, Gly, Asn Arg Arg,Gln, Lys, Glu, His Leu Ile, Phe, Tyr, Met, Val, Leu Pro Gly, Ala, Thr,Pro Thr Pro, Ser, Ala, Gly, His, Gln, Thr Ala Gly, Thr, Pro, Ala ValMet, Tyr, Phe, Ile, Leu, Val Gly Ala, Thr, Pro, Ser, Gly Ile Met, Tyr,Phe, Val, Leu, Ile Phe Trp, Met, Tyr, Ile, Val, Leu, Phe Tyr Trp, Met,Phe, Ile, Val, Leu, Tyr Cys Ser, Thr, Cys His Glu, Lys, Gln, Thr, Arg,His Gln Glu, Lys, Asn, His, Thr, Arg, Gln Asn Gln, Asp, Ser, Asn LysGlu, Gln, His, Arg, Lys Asp Glu, Asn, Asp Glu Asp, Lys, Asn, Gln, His,Arg, Glu Met Phe, Ile, Val, Leu, Met Trp Trp

TABLE 2 More Preferred Groups of Synonymous Amino Acids Amino AcidSynonymous Group Ser Ser Arg His, Lys, Arg Leu Leu, Ile, Phe, Met ProAla, Pro Thr Thr Ala Pro, Ala Val Val, Met, Ile Gly Gly Ile Ile, Met,Phe, Val, Leu Phe Met, Tyr, Ile, Leu, Phe Tyr Phe,Tyr Cys Cys, Ser HisHis, Gln, Arg Gln Glu, Gln, His Asn Asp, Asn Lys Lys, Arg Asp Asp, AsnGlu Glu, Gln Met Met, Phe, Ile, Val, Leu Trp Trp

TABLE 3 Most Preferred Groups of Synonymous Amino Acids Amino AcidSynonymous Group Ser Ser Arg Arg Leu Leu, Ile, Met Pro Pro Thr Thr AlaAla Val Val Gly Gly Ile Ile, Met, Leu Phe Phe Tyr Tyr Cys Cys, Ser HisHis Gln Gln Asn Asn Lys Lys Asp Asp Glu Glu Met Met, Ile, Leu Trp Met

Examples of production of amino acid substitutions in proteins which canbe used for obtaining muteins of IL-6 or IL-6R/IL-6 chimera, for use inthe present invention include any known method steps, such as presentedin U.S. Pat. Nos. 4,959,314, 4,588,585 and 4,737,462, to Mark et al;U.S. Pat. No. 5,116,943 to Koths et al., U.S. Pat. No. 4,965,195 toNamen et al; U.S. Pat. No. 4,879,111 to Chong et al; and U.S. Pat. No.5,017,691 to Lee et al; and lysine substituted proteins presented inU.S. Pat. No. 4,904,584 (Shaw et al).

Specific variants of IL-6 which are useful in connection with thepresent invention have been described (WO9403492A1). Furthermore,EP667872B1 describes mutant IL-6 with improved biological activity overwild type IL-6. In addition to this, EP656117B1 describes methods toisolate superagonists of IL-6. The mutants or superagnonists may be usedaccording to the invention.

The term “fused protein” refers to a polypeptide comprising IL-6 or anIL-6R/IL-6 chimera, or a variant or fragment thereof, fused with anotherprotein, which, e.g. has an extended residence time in body fluids. IL-6or an IL-6R/IL-6 chimera, may thus be fused to another protein,polypeptide or the like, e.g. an immunoglobulin or a fragment thereof.

“Functional derivatives” as used herein cover derivatives of IL-6 orIL-6R/IL-6 chimera, and their variants and fused proteins, which may beprepared from the functional groups which occur as side chains on theresidues or the N- or C-terminal groups, by means known in the art, andare included in the invention as long as they remain pharmaceuticallyacceptable, i.e. they do not destroy the activity of the protein whichis substantially similar to the activity of IL-6 or IL-6R/IL-6, and donot confer toxic properties on compositions containing it.

These derivatives may, for example, include polyethylene glycolside-chains, which may mask antigenic sites and extend the residence ofan IL-6R/IL-6 in body fluids. Other derivatives include aliphatic estersof the carboxyl groups, amides of the carboxyl groups by reaction withammonia or with primary or secondary amines, N-acyl derivatives of freeamino groups of the amino acid residues formed with acyl moieties (e.g.alkanoyl or carbocyclic aroyl groups) or O-acyl derivatives of freehydroxyl groups (for example that of seryl or threonyl residues) formedwith acyl moieties.

A “fragment” according to the present invention may e.g. be an activefraction of IL-6 or IL-6R/IL-6. The term fragment refers to any subsetof the molecule, that is, a shorter peptide which retains the desiredbiological activity, i.e. which has agonistic activity of gp130.Fragments may readily be prepared by removing amino adds from either endof the IL-6 or IL-6R/IL-6 molecule and testing the resultant fragmentfor its properties to bind to gp80 or gp130, respectively. Proteases forremoving one amino add at a time from either the N-terminal or the Cterminal of a polypeptide are known in the art, and so determiningfragments which retain the desired biological activity involves purelyroutine experimentation.

As fragments of IL-6 or an IL-6R/IL-6 chimera, variants and fusedproteins thereof, the present invention further covers any fragment orprecursors of the polypeptide chain of the protein molecule alone ortogether with associated molecules or residues linked thereto, e.g.sugar or phosphate residues, or aggregates of the protein molecule orthe sugar residues by themselves, provided said fraction has agonisticactivity on gp130, and in particular on gp130.

The term “salts” herein refers to both salts of carboxyl groups and toacid addition salts of amino groups of the IL-6 or an IL-6R/IL-6molecule or analogs thereof. Salts of a carboxyl group may be formed bymeans known in the art and include inorganic salts, for example, sodium,calcium, ammonium, ferric or zinc salts, and the like, and salts withorganic bases as those formed, for example, with amines, such astriethanolamine, arginine or lysine, piperidine, procaine and the like.Add addition salts include, for example, salts with mineral acids, suchas, for example, hydrochloric add or sulfuric acid, and salts withorganic acids, such as, for example, acetic acid or oxalic acid. Ofcourse, any such salt must retain the biological activity of IL-6 orIL-6R/IL-6 chimera, i.e., the ability to activate signaling throughgp130.

In a preferred embodiment of the invention, the substance of theinvention is glycosylated at one or more sites.

A glycosylated form of an IL-6R/IL-6 chimera has been described in WO99/02552 (PCT/IL98/00321), which is the chimeric molecule highlypreferred according to the invention. The IL-6R/IL-6 chimera describedtherein is a recombinant glycoprotein which was obtained fusing theentire coding sequence of the naturally-occurring soluble IL-6 receptorδ-Val (Novick et al., 1990) to the entire coding sequence of maturenaturally-occurring IL-6, both from human origin. The person skilled inthe art will appreciate that glycosylated IL-6 can be produced byrecombinant means as well, i.e. by expression in eukaryotic expressionsystems.

In accordance with the present invention, agonist may be produced in anyadequate eukaryotic or procaryotic cell type, like yeast cells, insectcells, bacteria, and the like. It is preferably produced in mammaliancells, most preferably in genetically engineered CHO cells as describedfor IL-6R/IL-6 in WO 99/02552. Whilst the protein from human origin ispreferred, it will be appreciated by the person skilled in the art thata similar fusion protein of any other origin may be used according tothe invention, as long as it retains the biological activity describedherein.

In a further embodiment of the invention, the substance of the inventionis not glycosylated. Advantageously, the chimeric molecule can then beproduced in bacterial cells, which are not capable of synthesizingglycosyl residues, but usually have a high yield of produced recombinantprotein. The production of nonglycosylated IL-6 has been described indetail in EP504751B1, for example.

In yet a further embodiment, the substance according to the inventioncomprises an immunoglobulin fusion, i.e. the molecules according to theinvention are fused to all or a portion of an immunoglobulin, and inparticular to an Fc fragment of an immunoglobulin. Methods for makingimmunoglobulin fusion proteins are well known in the art, such as theones described in WO 01/03737, for example. The person skilled in theart will understand that the resulting fusion protein of the inventionretains the biological activity of IL-6 or IL-6R/IL-6 chimera, i.e. thestimulation of gp130 signaling. The resulting fusion protein ideally hasimproved properties, such as an extended residence time in body fluids(half-life), increased specific activity, increased expression level, orfacilitated purification of the fusion protein.

Preferably, the substance of the invention is fused to the constantregion of an Ig molecule. It may be fused to heavy chain regions, likethe CH2 and CH3 domains of human IgG1, for example. Other isoforms of Igmolecules are also suitable for the generation of fusion proteinsaccording to the present invention, such as isoforms IgG₂ or IgG₄, orother Ig classes, like IgM or IgA, for example. Fusion proteins may thusbe monomeric or multimeric, hetero- or homomultimeric.

Functional derivatives of the substance of the invention may beconjugated to polymers in order to improve the properties of theprotein, such as the stability, half-life, bioavailability, tolerance bythe human body, or immunogenicity.

Therefore, a preferred embodiment of the invention relates to afunctional derivative of the substance of the invention comprising atleast one moiety attached to one or more functional groups which occuras one or more side chains on the amino acid residues.

A highly preferred embodiment relates to a substance of the inventionlinked to Polyethlyeneglycol (PEG). PEGylation may be carried out byknown methods, such as the ones described in WO 92/13095, for example.

Preferably, the substance signaling through gp130 is used in an amountranging from about 0.1 to 1000 μg/kg or about 1 to 500 μg/kg or lessthan about 100 μg/kg. It is further preferred to use the substancesignaling through gp130 in an amount of about 1 μg/kg or 3 μg/kg or 10μg/kg or 30 μg/kg.

In a preferred embodiment of the invention, the substance signalingthrough gp130 is administered daily. In a further preferred embodiment,the substance signaling through gp130 is administered three times perweek. In yet a further preferred embodiment, the substance signalingthrough gp130 is administered once a week.

The substance of the invention may be administered by any adequateroute. The subcutaneous route is highly preferred in accordance with thepresent invention.

The substance of the invention may be delivered to its site of action inany adequate formulation. Preferably, it may be delivered in form ofcells expressing and/or secreting IL-6, IL-6R/IL-6 chimera, a variant,fused protein or active fraction thereof. As illustrated in the examplesbelow, cells expressing and secreting IL-6R/IL-6 chimera in sufficientamounts have been generated by transfection into the cells using asuitable expression vector.

The invention therefore further relates to the use of a cell expressinga substance according to the invention, for manufacture of a medicamentfor the treatment and/or prevention of diabetic neuropathy. The cellsmay be administered in any suitable form. However, apolymer-encapsulated IL-6 or an IL-6R/IL-6 chimera expressing, andpreferably secreting cell, is a highly preferred mode of delivery ofIL-6R/IL-6 chimera. The encapsulation procedure is described in detaile.g. by Emerich et al (1994) or U.S. Pat. No. 5,853,385. Suitable celllines and stable expression systems are well known in the art.

The delivery of the substance according to the invention may also becarried out using a vector, such as an expression vector, comprising thecoding sequence of IL-6, an IL-6R/IL-6 chimera, a variant, fused proteinor fragment thereof. The vector comprises all regulatory sequencesneeded for expression of the desired protein in the human body, andpreferably in peripheral nervous cells. Regulatory sequences forexpression vectors are known by the person skilled in the art. Theinvention thus also relates to the use of a vector comprising the codingsequence of a substance according to the invention for manufacture of amedicament for the treatment and/or prevention of diabetic neuropathy.

Any expression vector known in the art may be used according to theinvention. However, the use of a virally derived gene therapy vector ishighly preferred.

The substance of the invention is preferably administered to the humanbody as a pharmaceutical composition. The pharmaceutical composition maycomprise the polypeptide of the invention as such, or cell expressingsaid polypeptide, or an expression vector, in particular a lentiviralgene therapy vector comprising the coding sequence of IL-6, anIL-6R/IL-6 chimera or a variant, fused protein, or active fragmentthereof, optionally together with one or more pharmaceuticallyacceptable carriers, diluents or excipients, for the treatment and/orprevention of diabetic neuropathy.

The definition of “pharmaceutically acceptable” is meant to encompassany carrier, which does not interfere with effectiveness of thebiological activity of the active ingredient and that is not toxic tothe host to which it is administered. For example, for parenteraladministration, the active component may be formulated in a unit dosageform for injection in vehicles such as saline, dextrose solution, serumalbumin and Ringer's solution.

The active component can be administered to a patient in a variety ofways. The routes of administration include intradermal, transdermal(e.g. in slow release formulations), intramuscular, intraperitoneal,intravenous, subcutaneous, oral, epidural, topical, and intranasalroutes. Any other therapeutically efficacious route of administrationcan be used, for example absorption through epithelial or endothelialtissues or by gene therapy wherein a DNA molecule is administered to thepatient (e.g. via a vector) which causes the active polypeptide to beexpressed and secreted in vivo. In addition the active molecule can beadministered together with other components of biologically activeagents such as pharmaceutically acceptable surfactants, excipients,carriers, diluents and vehicles.

For parenteral (e.g. intravenous, subcutaneous, intramuscular)administration, the active component can be formulated as a solution,suspension, emulsion or lyophilized powder in association with apharmaceutically acceptable parenteral vehicle (e.g. water, saline,dextrose solution) and additives that maintain isotonicity (e.g.mannitol) or chemical stability (e.g. preservatives and buffers). Theformulation is sterilized by commonly used techniques.

It is a further object of the present invention to provide for a methodfor treating and/or preventing diabetic neuropathy, comprisingadministering to a patient in need thereof an effective amount of asubstance which initiates signaling through the human gp130 receptor,optionally together with a pharmaceutically acceptable carrier.

An “effective amount” refers to an amount of the active ingredients thatis sufficient to affect the course and the severity of the diseasesdescribed above, leading to the reduction or remission of suchpathology. The effective amount will depend on the route ofadministration and the condition of the patient.

The dosage administered, as single or multiple doses, to an individualwill vary depending upon a variety of factor, including pharmacokineticproperties, the route of administration, patient conditions andcharacteristics (sex, age, body weight, health, size), extent ofsymptoms, concurrent treatments, frequency of treatment and the effectdesired. Adjustment and manipulation of established dosage ranges arewell within the ability of those skilled.

A method for treating diabetic neuropathy, comprising administering to apatient in need thereof an effective amount of a cell expressing IL-6 oran IL-6R/IL-6 chimera, or a variant, fused protein, active fractionthereof, is also considered in accordance with the present invention. Amethod for treating diabetic neuropathy comprising administering to apatient in need thereof an expression vector comprising the codingsequence of IL-6 or an IL-6R/IL-6 chimera, a variant, fused protein, oractive fraction thereof, is a further objects of the invention.

In a preferred embodiment of the invention, the expression vector is agene therapy vector. The use of a viral vector, in particular alentiviral vector, is highly preferred.

The present invention will now be described in more detail in thefollowing non-limiting examples and the accompanying drawings.

Having now fully described this invention, it will be appreciated bythose skilled in the art that the same can be performed within a widerange of equivalent parameters, concentrations and conditions withoutdeparting from the spirit and scope of the invention and without undueexperimentation.

While this invention has been described in connection with specificembodiments thereof, it will be understood that it is capable of furthermodifications. This application is intended to cover any variations,uses or adaptations of the invention following, in general, theprinciples of the invention and including such departures from thepresent disclosure as come within known or customary practice within theart to which the invention pertains and as may be applied to theessential features hereinbefore set forth as follows in the scope of theappended claims.

All references cited herein, including journal articles or abstracts,published or unpublished U.S. or foreign patent application, issued U.S.or foreign patents or any other references, are entirely incorporated byreference herein, including all data, tables, figures and text presentedin the cited references. Additionally, the entire contents of thereferences cited within the references cited herein are also entirelyincorporated by reference.

Reference to known method steps, conventional methods steps, knownmethods or conventional methods is not any way an admission that anyaspect, description or embodiment of the present invention is disclosed,taught or suggested in the relevant art.

The foregoing description of the specific embodiments will so fullyreveal the general nature of the invention that others can, by applyingknowledge within the skill of the art (including the contents of thereferences cited herein), readily modify and/or adapt for variousapplication such specific embodiments, without undue experimentation,without departing from the general concept of the present invention.Therefore, such adaptations and modifications are intended to be withinthe meaning an range of equivalents of the disclosed embodiments, basedon the teaching and guidance presented herein. It is to be understoodthat the phraseology or terminology herein is for the purpose ofdescription and not of limitation, such that the terminology orphraseology of the present specification is to be interpreted by theskilled artisan in light of the teachings and guidance presented herein,in combination with the knowledge of one of ordinary skill in the art.

EXAMPLES Example 1 Production of IL-6 and IL-6R/IL-6 Chimera in CHOCells

IL-6R/IL-6 Chimera

The cDNA sequences encoding for the soluble IL-6 receptor (natural formof sIL-6R found in urine, Oh et al., 1997) have been fused with thoseencoding for mature IL-6. Sequences for 3 bridging amino acids (EFM)were also present. The fused gene was inserted in an expression vectorunder the control of CMV promoter and introduced into CHO cells. Aproduction process has been developed and the resulting recombinantprotein has been purified by immunopurification using an anti-IL-6Rmonoclonal antibody. The purified IL-6 chimera has been shown to beglycosylated and to display an apparent MW of 85'000.

FIG. 10 schematically shows the composition of the IL-6R/IL-6 chimera.The mature protein comprises 524 amino acids.

A protein produced and purified as outlined above is suitable to beadministered according to the invention.

IL-6

Recombinant human IL-6 (r-hIL-6) is produced in genetically engineeredChinese Hamster Ovary (CHO) cells. The production process begins withthe growth and expansion of cells from a working cell bank (WCB) andcontinues under conditions where r-hIL-6 is secreted into the culturemedium. The r-hIL-6 harvested culture medium is purified byimmunochromatography using a specific anti-IL-6 monoclonal antibody(mAB). Further purification steps are used to yield a product with avery high level of purity.

r-hIL-6 is supplied as a sterile, freeze-dried preparation containingsuitable excipients. It is available in 2 different amounts, 35 μg and350 μg, and is reconstituted for use with water for injection. Thereconstitution volume for one vial of final formulated product isnormally 0.5 ml of water. The finished product should be stored in itsoriginal container at a temperature below 25° C.

The structure of r-hIL-6 has been confirmed by fast atomic bombardmentmass spectroscopy (FAB-MS), tryptic mapping and amino acid sequencing.FAB and electrospray mass spectroscopies were used to determine thecomposition and FAB and electrospray mass spectroscopies were used todetermine the composition and structure of the carbohydrate moieties ofIL-6. Residue aspargine-46 was identified as the N glycosylation siteand preliminary analysis of the N-linked carbohydrate showed that thedominant species were monosialyl fucosyl biantennary and disialylfucosyl biantennary structures. The O-glycosylation site was identifiedas either threonine −138 or −139.

Example 2 Effect of IL-6 in the Streptozotozin-Induced DiabeticNeuropathy Model Upon Intraperitoneal Administration

Materials and Methods

Animals

Six week-old male Sprague Dawley rats (Janvier, Le Genest-St-Isle,France) were distributed in 9 experimental groups (n=10) in accordancewith a randomization table: (a) a vehicle control group, injected with asterile solution of saline—BSA 0.02% (weight/volume); (b) a controlgroup consisting of animals injected with IL-6 at a dose of 100 μg/kgdissolved in a sterile solution of saline—BSA 0.02%; (c) astreptozotocin (STZ)-intoxicated group injected with a sterile solutionof saline—BSA 0.02%; (d) 5 treated, STZ-intoxicated groups consisting ofanimals receiving injections of IL-6 compound at 5 different doses: 1,3, 10, 30 and 100 μg/kg; (e) a STZ-intoxicated group and treated with areference compound: 4-methyl catechol (4-MC) at the dose of 10 μg/kg.

They were group-housed (2 animals per cage) and maintained in a roomwith controlled temperature (21-22° C.) and a reversed light-dark cycle(12 h/12 h) with food and water available ad libitum. All experimentswere carried out in accordance with institutional guidelines.

Induction of Diabetes and Pharmacological Treatment

Diabetes was induced by injection of a buffered solution ofstreptozotocin (Sigma, L'Isle d'Abeau Chesnes, France) in the surgicallydenuded left saphena magna, at a dose of 55 mg/kg body weight. The drugwas dissolved immediately before injection in 0.1 mol/l citrate bufferpH 4.5. The day of STZ injection was considered as Day (D) 0.

One week later, at D 10, tail vein blood was assayed for glycemia ineach individual animal using a glucometer (Glucotrend test, Roche,Mannheim, Germany). Animals showing a value below 260 mg/dl wereexcluded from the study. Glycemia was checked again at D 40, at the endof the experiment.

IP treatment (vehicle, IL-6 and 4-MC) was performed daily from D11 to D40.

Planning of Experiments

Body weight and survival rate were recorded every day.

Tail flick and EMG testings were performed once a week as followingtiming:

-   -   D-7: baseline (tail flick and EMG)    -   D 0: induction of diabetes by STZ injection    -   D 10: measure of glycemia    -   D 11: onset of the treatment (IL-6 and 4-MC)    -   D 25: EMG and tail flick testing    -   D 40: control of glycemia, EMG and tail flick tests, removal of        sciatic nerve

Sensitivity Test: Tall Flick

The tail of the rat was placed under a shutter-controlled lamp as a heatsource (Bioseb, Paris, France). The latency before the rat flicked itstail from the heat was recorded. A sensory alteration increases thelatency of flick. Two trials were performed and the mean value wascalculated and retained as characteristic value.

Electromyography

Electrophysiological recordings were performed using a Neuromatic 2000Melectromyograph (EMG) (Dantec, Les Ulis, France). Rats wereanaesthetized by intraperitoneal injection of 60 mg/kg ketaminechlorhydrate (Imalgene 500®, Rhône Mérieux, Lyon, France). The normalbody temperature was maintained at 30° C. with a heating lamp andcontrolled by a contact thermometer (Quick, Bioblock Scientific,Illkirch, France) placed on the tail surface.

Compound muscle action potential (CMAP) was recorded in thegastrocnemius muscle after stimulation of the sciatic nerve. A referenceelectrode and an active needle were placed in the hindpaw. A groundneedle was inserted on the lower back of the rat. Sciatic nerve wasstimulated with a single 0.2 ms pulse at a supramaximal intensity. Thevelocity of the motor wave was recorded and expressed in ms.

Sensitive nerve conduction velocity (SNCV) was also recorded. The tailskin electrodes were placed as follows: a reference needle inserted atthe base of the tail and an anoe needle placed 30 mm away from thereference needle towards the extremity of the tail. A ground needleelectrode was inserted between the anode and reference needles. Thecaudal nerve was stimulated with a series of 20 pulses (for 0.2 ms) atan intensity of 12.8 mA. The velocity was expressed in m/s.

Morphometric Analysis

Morphometric analysis was performed at the end of the study (D 40). Theanimals were anesthetized by IP injection of 100 mg/kg Imalgene 500®. A5 mm-segment of sciatic nerve was excised for histology. The tissue wasfixed overnight with 4% glutaraldehyde (Sigma, L'Isle d'Abeau-Chesnes,France) solution in phosphate buffer solution (pH=7.4) and maintained in30% sucrose at +4° C. until use. The nerve sample was fixed in 2% osmiumtetroxide (Sigma, L'Isle d'Abeau-Chesnes, France) solution in phosphatebuffer solution for 2 h., dehydrated in serial alcohol solution, andembedded in Epon. Embedded tissues were then placed at +70° C. during 3days of polymerization. Transverse sections of 1.5 μm were cut with amicrotome, stained with a 1% toluidine blue solution (Sigma, L'Isled'Abeau-Chesnes, France) for 2 min, dehydrated and mounted in Eukitt.Twenty sections per sample were examined using an optical microscope(Nikon, Tokyo, Japan) and 6 randomly selected slices were analyzed usinga semi-automated digital image analysis software (Biocom, France). Tworandomly selected fields per slice were studied. The followingparameters were calculated: (a) fiber diameter, (b) axon diameter, (c)myelin thickness (see below).

For counting the total number of fibers per nerve section, 3 randomizedslices per sample were selected and 2 fields per slice were analyzed.

Data Analysis

Global analysis of the data was performed using one factor or repeatedmeasure analysis of variance (ANOVA) and one way ANOVA Dunnett's testwas used when anova test indicated a significant difference. No post-hocanalyses were performed. The level of significance was set at p<0.05.Results are expressed as mean±standard error of the mean (s.e.m.).

Results

Animal Weight

As illustrated in FIG. 1, a significant intergroup difference in bodyweight evolution was noted in this study [f (8, 296)=19.47 and p<0.001;repeated measure ANOVA]. From d 5 to d 40, theSTZ-intoxicated/IL-6-treated animals displayed a significant decrease inbody weight (p<0.05; one way ANOVA and p<0.05 control versus (vs) STZ;control/il-6 (100 μg/kg) vs STZ; control vs STZ+il-6 and control/il-6(100 μg/kg) vs STZ+IL-6; Dunnett's test).

Diabetic animals treated with IL-6 at a dose of 10 μg/kg displayed abody weight significantly higher than that of other doses of il-6 [f (5,185)=1.16 and p=0.08 repeated measures ANOVA].

It could be noted that the STZ-intoxicated/IL-6 treated animals at thedose of 100 μg/kg displayed a body weight decreased throughout the study(starting at the beginning of the treatment).

Glycemia

FIG. 2 a shows that at d 10 control animals presented a glycemia valueequal at 100 mg/dl. On the other hand, STZ-intoxicated rats displayed aplasma glucose concentration higher than 260 mg/dl and were consideredas diabetic.

FIG. 2 b shows that STZ-intoxicated rats were still diabetic at d 40.

Sensitivity Test: Tail Flick

There was a significant intergroup difference in the evolution oftail-flick test performances [F (8, 16)=2.07 and p=0.013; repeatedmeasure ANOVA] (FIG. 3). The latency before the rats flicked their tailfrom the heat was significantly increased in diabetic non treatedanimals (control/vehicle vs STZ/vehicle p<0.001; Dunnett's test). In theD 25 and D 40, the reaction time was not increased in the animalstreated with IL-6 at doses of 1, 3, 10 and 100 μg/kg and treated with4-MC at 10 μg/kg. Indeed, no significant difference between these groupswas found (p>0.05; Dunnett's test).

Electrophysiological Measurements

Latency of the Compound Muscle Action Potential

There was a significant difference between the groups in the latency ofthe CMAP throughout the study [F (8, 16)=5.901 and p<0.001; repeatedmeasures ANOVA]. The latency was significantly increased in diabeticuntreated rats (on days 25 and 40: p<0.001; one way ANOVA). Thisincrease was less important in IL-6-treated groups; especially for theIL-6 (10 μg/kg)-treated group which presented, on days 25 and 40, nosignificant difference with the latency value of the control/vehiclegroup (FIG. 4).

Moreover, on day 25, each IL-6 treated/STZ groups displayed a CMAPlatency significantly shorter than the CMAP latency of the vehicle/STZgroup (p=0.001, Dunnett's test).

On day 40, the same conclusion was drawn.

A significant difference was seen between IL-6 treated/STZ animals (10mg/kg) and the 4 other IL-6 treated groups (1, 3, 10, 30 and 100 μg/kg)(D 25: p=0.002, D 40: p=0.003; one way ANOVA test). The 3 μg/kg and 10μg/kg treated animals displayed a lower significantly latency than the1, 30 and 100 μg/kg IL-6 treated/STZ groups (p<0.05; Dunnett's test) onday 25 as well as day 40.

Sensory Nerve Conduction Velocity

A significant difference was noted between the groups in the SNCVthroughout the study [F (8,16)=5.518 and p<0.001; repeated measuresANOVA] (FIG. 5). Diabetic rats displayed a significant decrease of theSNCV (on days 25 and 40: p<0.001; one way ANOVA) in contrast with thecontrol/vehicle group. Moreover, on day 25 no significant difference wasobserved between the control/vehicle group and the 10 μg/kg IL-6-treatedgroup (p=0.426; Dunnett's test), whereas a significant difference wasseen between all the other groups. On day 25, only the 10 and the 30μg/kg displayed a significant difference with the STZ/vehicle group (10μg/kg vs vehicle STZ groups: p<0.001, 30 μg/ml vs STZ groups, p=0.004,Dunnett's test). On day 40, the animal treated with 10 μg/kg did notshow any significant difference with the STZ/vehicle treated animalshowever, the SNCV value for this group was higher than the otherSTZ/IL-6 treated animals.

It could be noted that the SNCV gradually increased throughout the studyin the control/vehicle animals due to a normal maturation of theperipheral nerve structure (Gao et al., 1995, Malone et al., 1996).

Morphometric Analysis

Axon Diameter

A significant intergroup difference was found in the axon diameter(p<0.001; one way ANOVA) (FIG. 6). Vehicle/STZ animals displayed asignificant decrease in the axon diameter in comparison with the controlrats (p=0.08; Dunnett's test). Treatment with IL-6 reversed thisdecrease of axon diameter, since the dose of 3 μg/kg (IL-6-treated ratsvs STZ/vehicle p<0.001; Dunnett's test). Moreover, a significantdifference was noted between the control group and the control/IL-6 (100μg/kg) group (p<0.001; Dunnett's test). No significant difference wasfound between the control/vehicle group and the 4-MC-treated group(p=0.657; Dunnett's test).

Fiber Diameter

FIG. 7 shows that there was a significant difference between the 9groups in fiber diameter (p<0.001, one way ANOVA). The STZadministration leads to a significant decrease of fiber diameter(control/vehicle vs STZ/vehicle p=0.005; Dunnett's test). A significantdifference was observed between vehicle/STZ rats and IL-6-treated/STZrats (p<0.005; Dunnett's test). The IL-6-treated animals displayed alarger fiber diameter than vehicle/STZ animals. Moreover, a significantdifference was found between the control group and the control/IL-6 (100μg/kg) group (p<0.001; Dunnett's test). The animals treated with 4-MC atthe dose of 10 μg/kg presented no significant difference with thecontrol/vehicle group (p=0.628; Dunnett's test).

Myelin Thickness

Comparison of the myelin thickness revealed a significant differencebetween the 9 groups (p<0.001; one way ANOVA) (FIG. 8). The myelinthickness was significantly smaller in vehicle/STZ animals thancontrol/vehicle and IL-6-treated animals (3, 10, 30 and 100 μg/kg)(p<0.01; Dunnett's test). It could be noted that all the IL-6treated/STZ animals presented a myelin thickness significantly higherthan the vehicle/STZ group. Moreover, we noted a significant differencebetween control/vehicle and control/IL-6 (100 μg/kg) groups (p<0.001;Dunnett's test).

Total Number of Myelinated Fibers

As shown in FIG. 9, there was a significant intergroup difference intotal number of myelinated fibers (p<0.001; one way ANOVA). Thevehicle/STZ animals displayed a smaller number of fibers than thecontrol animals (p<0.001; Dunnett's test). By contrast, theIL-6-treated/STZ animals had an increased number of fibers as comparedwith the vehicle/STZ animals (p<0.001; Dunnett's test). TheSTZ-intoxicated animals treated with 4-MC had a total number of fibersanalogous to those of the control animals. Moreover, no significantdifference between control/vehicle group and control/IL-6 (100 μg/kg)group was noted.

Conclusion

In this study, animals intoxicated with the streptozotocin and whichdevelop a diabetes several days later, have been used as model ofinduced-neuropathy. The animal becomes diabetic 3-4 days after theinduction.

The diabetic animals have been treated by different doses of IL-6 (1, 3,10, 30 and 100 μg/kg) on 30 days chronically. The treatment has beenadministrated intraperitoneally every day starting 10 days after theinduction until the sacrifice of the animal 40 days after theSTZ-induction. This treatment could be considered as a curativetreatment in that IL-6 has been administered after the first moleculardamages caused by a prolonged hyperglycemia.

The present protocol shows that a IL-6 treatment of 30 days induces aneuroprotection against the diabetic neuropathy. The behavioral analyseswith tail flick and the EMG testing (sensory and motor velocities) showthe neuroprotective effect of IL-6 especially for the doses of 3 and 10μg/kg.

The low doses as well as the high concentrations displayedneuroprotective effect. Indeed, the more the doses of treatmentincrease, the less the neuroprotective effect is significant. Moreover,the highest dose (100 μg/kg) does not display a pronounced effect andseems to have a toxic effect on the general behavior of the STZ-animals.The control animals (not treated with STZ) treated with IL-6 at 100μg/kg were more excited than the vehicle control animals or the STZ ratstreated with low concentrations of IL-6. Moreover, these animals (IL-6100 μg/kg) were difficult to manipulate for the experimentator. The samewas observed in the STZ/IL-6 100 μg/kg. Nevertheless, these animalsseemed less excited (probably due to their weakness due to their largeloss of body weight).

The neuroprotective effect is focused on the sensory fibers as well asmotor fibers (the CMAP velocity was not altered with a IL-6 treatment).

Concerning the morphological analysis, the neuroprotection induced bythe IL-6 treatment is very clear for all studied doses. The fibers ofthe STZ/vehicle animals displayed a decrease of the myelin sheath and analteration of the axon, which finally induce a degeneration of thefibers (shown with a decrease of the total number of fibers).

It was demonstrated in this study that the treatment with IL-6(especially 10 μg/kg) protected the myelin sheath and the axonaldegeneration.

It must be noted that the high dose of IL-6 (100 μg/kg) induces anharmful effect on the fibers in healthy animals. Indeed, the fibers seemto suffer, there is no loss of fiber but the sheath and the generalaspect of the fibers are altered. Whereas this effect is not recorded inthe diabetic animals treated with this large dose of IL-6 (the toxiceffect is mainly focused on the general behavior of the animalcharacterized by a large decrease of the body weight).

In conclusion, IL-6 induces a dear neuroprotective effect after achronic treatment of 30 days as well as on sensory than motor fibers,probably acting by a direct effect on the fiber and reducing theinflammatory process of neurodegeneration.

Example 3 Effect of IL-6 in the Streptozotozin-Induced DiabeticNeuropathy Model Upon Subcutaneous Administration

The aim of this study was to evaluate the effect of the IL-6 via thesubcutaneous route at different dosage and timing in the same model ofneuropathy.

Animals

Study A

Six week-old male Sprague Dawley rats (Janvier, Le Genest-St-Isle,France) were distributed in 6 experimental groups in accordance with arandomization table: (a) a vehicle control group (n=4), injected with asterile solution of saline—BSA 0.02% (weight/volume); (b) astreptozotocin (STZ)-intoxicated group (n=10) injected with a sterilesolution of saline—BSA 0.02%; (c) 4 treated, STZ-intoxicated groups(n=10) consisting of animals receiving daily SC injections of IL-6compound at 4 different doses: 1, 3, 10, 30 μg/kg.

They were group-housed (2 animals per cage) and maintained in a roomwith controlled temperature (21-22° C.) and a reversed light-dark cycle(12 h/12 h) with food and water available ad libitum. All experimentswere carried out in accordance with institutional guidelines.

Study B

Six week-old male Sprague Dawley rats (Janvier, Le Genest-St-Isle,France) were distributed in 7 experimental groups in accordance with arandomization table: (a) a vehicle control group (n=4), injected with asterile solution of saline—BSA 0.02% (weight/volume); (b) astreptozotocin (STZ)-intoxicated group (n=10) injected with a sterilesolution of saline—BSA 0.02%; (c) 4 treated, STZ-intoxicated groups(n=10) consisting of animals receiving SC injections of IL-6 compound 3times per week at 4 different doses: 1, 3, 10, 30 μg/kg; (d) a treated,STZ-intoxicated group (n=10) consisting of animals receiving IPinjections of IL-6 compound at 10 μg/kg.

They were group-housed (2 animals per cage) and maintained in a roomwith controlled temperature (21-22° C.) and a reversed light-dark cycle(12 h/12 h) with food and water available ad libitum. All experimentswere carried out in accordance with institutional guidelines.

Study C

Six week-old male Sprague Dawley rats (Janvier, Le Genest-St-Isle,France) were distributed in 6 experimental groups in accordance with arandomization table: (a) a vehicle control group (n=4), injected with asterile solution of saline—BSA 0.02% (weight/volume); (b) astreptozotocin (STZ)-intoxicated group (n=10) injected with a sterilesolution of saline—BSA 0.02%; (c) 4 treated, STZ-intoxicated groups(n=10) consisting of animals receiving SC injections of IL-6 compoundonce a week at 4 different doses: 1, 3, 10, 30 μg/kg.

They were group-housed (2 animals per cage) and maintained in a roomwith controlled temperature (21-22° C.) and a reversed light-dark cycle(12 h/12 h) with food and water available ad libitum. All experimentswere carried out in accordance with institutional guidelines.

Induction of Diabetes and Pharmacological Treatment

Diabetes was induced by injection of a buffered solution ofstreptozotocin (Sigma, L'Isle d'Abeau Chesnes, France) in the surgicallydenuded left saphena magna, at a dose of 55 mg/kg body weight. The drugwas dissolved immediately before injection in 0.1 mol/l citrate bufferpH 4.5. The day of STZ injection was considered as Day (D) 0.

One week later, at D 10, tail vein blood was assayed for glycemia ineach individual animal using a glucometer (Glucotrend test, Roche,Mannheim, Germany). Animals showing a value below 260 mg/dl wereexcluded from the study. Glycemia was checked again at D 40, at the endof the experiment.

Treatment (vehicle and IL-6) was performed from D11 to D 40.

Planning of Experiments

Body weight and survival rate were recorded every day.

Tail flick and EMG testings were performed once a week as followingtiming:

-   -   D-7: baseline (tail flick, locomotor activity and EMG)    -   D 0: induction of diabetes by STZ injection    -   D 10: measure of glycemia    -   D 11: onset of the treatment (IL-6)    -   D 24: tail flick test    -   D 25: locomotor activity in OF (open field)    -   D 26: EMG testing    -   D 38: tail flick test    -   D 39: locomotor activity in OF    -   D 40: control of glycemia, EMG testing, removal of sciatic nerve

Sensitivity Test: Tail Flick

The tail of the rat was placed under a shutter-controlled lamp as a heatsource (Bioseb, Paris, France). The latency before the rat flicked itstail from the heat was recorded. A sensory alteration increases thelatency of flick. Two trials were performed and the mean value wascalculated and retained as characteristic value.

Locomotor Activity in Open Field

The animal was placed in a Plexiglas (80×80×40 cm) open field (OF). Thefloor was divided into 16 equal squares. For each animal, thespontaneous locomotor activity and the number of rearings were recordedduring a 10 min period.

Electromyography

Electrophysiological recordings were performed using a Neuromatic 2000Melectromyograph (EMG) (Dantec, Les Ulis, France). Rats wereanaesthetized by intraperitoneal injection of 60 mg/kg ketaminechlorhydrate (Imalgene 500@, Rhone Mérieux, Lyon, France). The normalbody temperature was maintained at 30° C. with a heating lamp andcontrolled by a contact thermometer (Quick, Bioblock Scientific,Illkirch, France) placed on the tail surface.

Compound muscle action potential (CMAP) was recorded in thegastrocnemius muscle after stimulation of the sciatic nerve. A referenceelectrode and an active needle were placed in the hindpaw. A groundneedle was inserted on the lower back of the rat. Sciatic nerve wasstimulated with a single 0.2 ms pulse at a supramaximal intensity. Thevelocity of the motor wave was recorded and expressed in ms.

Sensitive nerve conduction velocity (SNCV) was also recorded. The tailskin electrodes were placed as follows: a reference needle inserted atthe base of the tail and an anode needle placed 30 mm away from thereference needle towards the extremity of the tail. A ground needleelectrode was inserted between the anode and reference needles. Thecaudal nerve was stimulated with a series of 20 pulses (for 0.2 ms) atan intensity of 12.8 mA. The velocity was expressed in m/s.

Morphometric Analysis

Morphometric analysis was performed at the end of the study (D 40). Theanimals were anesthetized by IP injection of 100 mg/kg Imalgene 500®. A5 mm-segment of sciatic nerve was excised for histology. The tissue wasfixed overnight with 4% glutaraldehyde (Sigma, L'Isle d'Abeau-Chesnes,France) solution in phosphate buffer solution (pH=7.4) and maintained in30% sucrose at +4° C. until use. The nerve sample was fixed in 2% osmiumtetroxide (Sigma, L'Isle d'Abeau-Chesnes, France) solution in phosphatebuffer solution for 2 h., dehydrated in serial alcohol solution, andembedded in Epon. Embedded tissues were then placed at +70° C. during 3days of polymerization. Transverse sections of 1.5 μm were cut with amicrotome, stained with a 1% toluidine blue solution (Sigma, L'Isled'Abeau-Chesnes, France) for 2 min, dehydrated and mounted in Eukitt.Twenty sections per sample were examined using an optical microscope(Nikon, Tokyo, Japan) and 6 randomly selected slices were analyzed usinga semi-automated digital image analysis software (Biocom, France). Tworandomly selected fields per slice were studied. The followingparameters were calculated: (a) fiber diameter, (b) axon diameter, (c)myelin thickness.

For counting the total number of fibers per nerve section, 3 randomizedslices per sample were selected and 2 fields per slice were analyzed.

Data Analysis

Global analysis of the data was performed using one factor or repeatedmeasure analysis of variance (ANOVA) and one way ANOVA. Dunnett's testwas used when anova test indicated a significant difference. No post-hocanalyses were performed. The level of significance was set at p<0.05.Results are expressed as mean±standard error of the mean (s.e.m.).

Results

Study A

Animal Weight

As illustrated in FIG. 11, a significant intergroup difference in bodyweight evolution was noted in this study [f (5, 185)=9.20 and p<0.001;repeated measure anova]. from d 5 to d 40, theSTZ-intoxicated/IL-6-treated animals displayed a significant decrease inbody weight (p<0.05; one way ANOVA and p<0.05 control versus (vs) STZ;control vs STZ+il-6; dunnett's test).

Diabetic animals treated with il-6 at a dose of 10 μg/kg displayed abody weight significantly higher than that of other doses of il-6 [f (4,148)=2.93 and p<0.001 repeated measures ANOVA].

Glycemia

FIG. 12 shows that at d 10 control animals presented a glycemia valueequal at 120 mg/dl. On the other hand, STZ-intoxicated rats displayed aplasma glucose concentration higher than 260 mg/dl and were consideredas diabetic.

It was noted that STZ-intoxicated rats were still diabetic at d 41 (therat no 2 of the stz/IL-6 (10 μg/kg) group has been eliminated of thestudy because of his glycemia below 260 mg/dl).

Sensitivity Test: Tail Flick

There was no significant intergroup difference in the evolution oftail-flick test performances [F (5, 10)=1.81 and p=0.072; repeatedmeasure ANOVA] (FIG. 13). Nevertheless, the latency before the ratsflicked their tail from the heat was increased in diabetic non treatedand treated with IL-6 at higher doses animals. In the D 38, the reactiontime was not increased in the animals treated with IL-6 at doses of 1and 3 μg/kg. Indeed, no significant difference between these groups wasfound (p>0.05; Dunnett's test).

Locomotor Activity in OF

As shown in FIGS. 14A and 14B, there was a significant differencebetween the groups in the number of crossed squares and rearingsthroughout the study [respectively, F (5, 10)=5.99 with p<0.001 and F(5, 10)=4.22 with p<0.001; repeated measures ANOVA]. On day 25 and 40,diabetic, treated or not, displayed a lower locomotor activity which wascharacterized by a significant decrease of numbers of crossed squaresand rearings (control vs STZ/vehicle and control vs STZ/IL-6 p<0.01Dunnett's test).

It could be noted that the animals treated with the doses of 1 and 3μg/kg presented a higher locomotor activity than the STZ/vehicle rats.

Electrophysiological Measurements

Latency of the Compound Muscle Action Potential

There was a significant difference between the groups in the latency ofthe CMAP throughout the study [F (5, 10)=5.71 and p<0.001; repeatedmeasures ANOVA]. The latency was significantly increased in diabeticuntreated rats (on days 26 and 41 p<0.01; one way ANOVA). Moreover, thisincrease was less important in each IL-6-treated groups (FIG. 15).

On days 26 and 41, each STZ/IL-6-treated groups displayed a CMAP latencysignificantly shorter than the CMAP latency of the STZ/vehicle group(p=0.05, Dunnett's test).

Sensory Nerve Conduction Velocity

A significant difference was noted between the groups in the SNCVthroughout the study [F (5,10)=3.78 and p<0.001; repeated measuresANOVA] (FIG. 16). Diabetic rats displayed a significant decrease of theSNCV (on days 26 and 41: p<0.01; one way ANOVA) in contrast with thecontrol/vehicle group.

On days 26 and 41, all the IL-6 treated animals displayed a significantdifference with the STZ/vehicle group (p<0.05; Dunnett's test).Moreover, on day 41 no significant difference was observed between thecontrol/vehicle group and the 3 and 30 μg/kg IL-6-treated groups(respectively, p=0.054 and p=0.184; Dunnett's test), whereas asignificant difference was seen between all the other groups.

Morphometric Analysis

Fiber Diameter

As shown in FIG. 17, a significant intergroup difference was seen infiber diameter (p<0.001; one way ANOVA). A decrease of fiber diameterwas observed in diabetic rats in comparison with control/vehicle group(p<0.001; Dunnett's test). Moreover, IL-6 treatment, for all testeddoses significantly prevents from this diameter decrease (STZ/vehicle vsSTZ/IL-6 treated: p<0.05; Dunnett's test).

Axon Diameter

There was a significant difference between groups in axon diameter(p<0.001 one way ANOVA) (FIG. 18). STZ/vehicle group displayed asignificant decrease of axon diameter (control/vehicle vs STZ/vehicle:p<0.001; Dunnett's test). Animals treated with IL-6 at all tested dosespresented an axon diameter significantly higher than diabetic nontreated rats (p<0.05; Dunnett's test).

Myelin Thickness

A significant intergroup difference was found in myelin thickness(p<0.001; one way ANOVA) (FIG. 19). A significant decrease of myelinthickness was observed in diabetic rats in comparison withcontrol/vehicle animals (p<0.001; Dunnett's test). Moreover, thisdecrease was significantly less important in IL-6-treated groups(p<0.05; Dunnett's test).

Percentage of Degenerate Fibers

As shown in FIGS. 20 and 21, diabetic and no-treated rats dispalyed asignificant decrease of myelinated fibers (p<0.001; one way ANOVA).Treatment with IL-6 at doses of 3, 10 and 30 μg/kg every day, decreasedsignificantly the percentage of degenerate fibers in comparison withSTZ/vehicle group (p<0.001; Dunnett's test).

Study B

Animal Weight

There was a significant intergroup difference in body weight evolution[F (6, 168)=9.24 and p<0.001; repeated measures ANOVA] (FIG. 22). From D5 to D 40, the STZ-intoxicated animals displayed a significant decreasein body weight (p<0.05; one way ANOVA and p<0.001 control/vehicle vsSTZ; control/vehicle vs STZ-IL-6-treated groups; Dunnett's test).

No significant difference was found between the STZ/IL-6-treated groupsin the body weight from D 5 to D 40 [F (5, 125)=1.08 and p=0.26;repeated measures ANOVA].

Glycemia

As illustrated in FIG. 23, at D 10 STZ-intoxicated rats displayed aplasma glucose concentration higher than 260 mg/dl whereas controlanimals presented a glycemia value around 100 mg/dl.

Moreover, at D 40 STZ-intoxicated rats were still diabetic, indeed theirglycemia was higher than 500 mg/dl.

Sensitivity Test: Tail Flick

There was a significant intergroup difference in the evolution of tailflick test performances [F (6, 12)=2.13 and p=0.02; repeated measuresANOVA] (FIG. 24). At days 24 and 38, diabetic and non-treated ratsdisplayed a significant increased reaction time in comparison with thecontrol/vehicle and STZ/IL-6-treated groups (p<0.05 Dunnett's test).

Moreover, the reaction time was less increased in the animals treatedwith IL-6 at doses of 10 and 30 μg/kg.

Locomotor Activity in OF

As shown in FIGS. 25A and 25B, there was a significant differencebetween the groups in the number of crossed squares and rearingsthroughout the study [respectively, F (5, 10)=with p<0.001 and F (5,10)=with p<0.001; repeated measures ANOVA]. On day 25 and 40, diabetic,treated or not, displayed a lower locomotor activity which wascharacterized by a significant decrease of numbers of crossed squaresand rearings (control vs STZ/vehicle and control vs STZ/IL-6 p<0.05;Dunnett's test).

Electrophysiological Measurements

Latency of the Compound Muscle Action Potential

As shown in FIG. 26, there was a significant difference between thegroups in the latency of the CMAP throughout the study [F (6, 12)=3.97and p<0.001; repeated measures ANOVA]. On D 26 and D 40, a significantincrease of the latency was observed in the diabetic non-treated ratsand the animals treated with IL-6 at low dose (p<0.001; one way ANOVA).Moreover, no significant difference was found between thecontrol/vehicle and STZ/IL-6 treated (10 and 30 μg/kg) groups (p>0.05;one way ANOVA).

Sensory Nerve Conduction Velocity

There was a significant difference between the groups in the SNCVmeasure throughout the study [F (6, 12)=3.38 and p<0.001; repeatedmeasures ANOVA] (FIG. 17). Since D 26, diabetic and treated or not ratsdisplayed a decrease of the SNCV (D 26: p<0.001 and D 40: p<0.001; oneway ANOVA).

On D 26, the control/vehicle and STZ/IL-6 treated animals displayed aSNCV significantly higher than that of STZ/vehicle rats (p<0.05;Dunnett's test).

On D 40, The rats treated with IL-6 at higher doses presented a value ofSNCV more important than that of STZ/vehicle group.

Morphometric Analysis

Fiber Diameter

As shown in FIG. 28, a significant intergroup difference was noted infiber diameter (p=0.045; one way ANOVA). STZ-intoxicated animalsdisplayed a significant decrease of fiber diameter in comparison withcontrol/vehicle rats (p<0.05; Dunnett's test). Daily IP treatment withIL-6 prevented from this fiber diameter decrease (STZ/vehicle vsSTZ/IL-6 IP: p=0.026; Dunnett's test).

Axon Diameter

There was a significant difference between groups in axon diameter(p=0.034; one way ANOVA) (FIG. 29). A significant decrease of axondiameter was observed in STZ-intoxicated animals (p<0.05; Dunnett'stest). Rats treated with IL-6 at 10 μg/kg by IP route displayed asignificant difference with STZ/vehicle animals (p=0.045 Dunnett'stest).

Myelin Thickness

A significant intergroup was found in myelin thickness (p=0.05; one wayANOVA) (FIG. 30). STZ-intoxicated animals displayed a significantdecrease of myelin thickness (p<0.05; Dunnett's test). A daily IPtreatment with IL-6 prevented from this decrease of myelin thickness(STZ/vehicle vs STZ/IL-6 IP: p<0.005; Dunnett's test).

Percentage of Degenerate Fibers

As illustrated in FIGS. 31 and 32, a significant intergroup differencein percentage of degenerate fibers was found (p<0.001; one way ANOVA).This percentage was significantly higher in STZ/vehicle group thancontrol/vehicle one (p<0.001; Dunnett's test). Percentage wassignificantly decreased in IL-6-treated animals (STZ/vehicle vsSTZ/IL-6: p<0.001; Dunnett's test).

Study C

Animal Weight

As illustrated in FIG. 33, a significant intergroup difference in bodyweight evolution was observed throughout the study [F (5, 145)=15.46 andp<0.001 repeated measures ANOVA]. From D 5 to D 40, the STZ-intoxicatedanimals displayed a significant decrease in body weight (p<0.001; oneway ANOVA and p<0.001 control/vehicle vs STZ; control/vehicle vsSTZ-IL-6-treated groups; Dunnett's test).

Moreover, the animals treated with IL-6 at 30 μg/kg displayed a decreaseof body weight significantly less important than the others IL-6 treatedgroups [F (4, 104)=2.17 and p<0.001; repeated measures ANOVA].

Glycemia

FIG. 34 shows that control/vehicle group presented a glycemia valueinferior at 120 mg/dl on Dl 0 and D 40. On the other hand,STZ-intoxicated rats displayed a plasma glucose concentration higherthan 260 mg/dl, so they were considered as diabetic on D 10 and D 40.

Sensitivity Test: Tail Flick

There was a significant intergroup difference in the reaction timethroughout the study [F (5, 10)=2.30 and p=0.02; repeated measuresANOVA] (FIG. 35). The latency before the rats flicked their tail fromthe heat was significantly increased in STZ/vehicle animals (on D 24 andD 38: p<0.005; one way ANOVA). On D 24, there was no significantdifference in the latency of reaction between the control/vehicle andSTZ/IL-6 (30 μg/kg) groups (p=0.31; Dunnett's test). On D 38, the ratstreated with IL-6 at 10 and 30 μg/kg displayed a reaction time similarto control one (p=0.3; Dunnett's test).

Locomotor Activity in OF

There was a significant intergroup difference in the number of crossedsquares and rearings throughout the study (respectively [F (5, 10)=withp<and F (5, 10) 2.15 with p=0.028; repeated measures ANOVA) (FIGS. 26Aand 26B).

On D 25 and D 39, STZ-intoxicated, treated or not, animals displayed asignificant lower locomotor activity than the control rats one (p<0.05;Dunnett's test). This difference was noted in the mean number of crossedsquares and the number of rearings throughout the recorded period.Nevertheless, the group of animals treated with IL-6 at the dose of 30μg/kg presented a larger locomotor activity than STZ/vehicle group.

Electrophysiological Measurements

Latency of the Compound Muscle Action Potential

No significant inter group difference was noted in the latency of theCMAP throughout the study [F (5, 10)=1.33 and p=0.23; repeated measuresANOVA] (FIG. 27). Nevertheless, a significant difference between thegroups was observed on D 26 and D 40 (p<0.05; one way ANOVA).STZ-intoxicated rats displayed an increased latency in comparison withthe control/vehicle group. Moreover, this increase was less important ingroups treated with IL-6 at high doses, indeed a significant differencewas noted between STZ/vehicle and STZ/IL-6 groups (on D 26: STZ/vehiclevs STZ/IL-6 at 3, 10, 30 μg/kg: p<0.005 and on D 40: STZ/vehicle vsSTZ/IL-6 at 30 μg/kg: p<0.005; Dunnett's test).

Sensory Nerve Conduction Velocity

As illustrated in FIG. 38, a significant intergroup difference was notedin the sensore nerve conduction velocity throughout the study [F (5,10)=2.18 and p=0.025; repeated measures ANOVA]. A significant decreaseof velocity was observed in STZ-intoxicated animals (control/vehicle vsSTZ/vehicle and STZ/IL-6 groups: p<0.05; Dunnett's test). In addition,treatment with IL-6 at 10 and 30 μg/kg lead to a velocoty loss lessimportant in diabetic animals (on D 26, STZ/IL-6 at 10 and 30 μg/kg vsSTZ/vehicle: p<0.05; Dunnet's test).

Morphometric Analysis

Fiber Diameter

As shown in FIG. 39, a significant intergroup difference was noted infiber diameter (p<0.001; one way ANOVA). STZ-intoxicated animalsdisplayed a significant decrease of fiber diameter in comparison withcontrol/vehicle rats (p<0.005; Dunnett's test). IL-6 treated animals (atall doses) presented a significantly higher diameter fiber thanSTZ/vehicle rats (p<0.05; Dunnett's test).

Axon Diameter

There was a significant difference between groups in axon diameter(p<0.001 one way ANOVA) (FIG. 40). A significant decrease of axondiameter was observed in STZ-intoxicated animals (p<0.01; Dunnett'stest). Rats treated with IL-6 at 1, 3, 10 and 30 μg/kg displayed asignificant difference with STZ/vehicle animals (p<0.05; Dunnett'stest).

Myelin Thickness

A significant intergroup was found in myelin thickness (p<0.001; one wayANOVA) (FIG. 41). STZ-intoxicated animals displayed a significantdecrease of myelin thickness (p<0.001; Dunnett's test). A daily SCtreatment with IL-6 prevented from this decrease of myelin thickness(STZ/vehicle vs STZ/IL-6 1 and 30 μg/kg: p<0.005 Dunnett's test).

Percentage of Degenerate Fibers

As illustrated in FIGS. 42 and 43, a significant intergroup differencein percentage of functional myelinated fibers was found (p<0.001; oneway ANOVA). This percentage was significantly lower in STZ/vehicle groupthan control/vehicle one (p<0.001; Dunnett's test). Percentage wassignificantly increased in animals treated with IL-6 at 3, 10 and 30μg/kg (p<0.001; Dunnett's test).

Conclusion

In this study, animals intoxicated with the streptozotocin and whichdevelop a diabetes several days later, have been used as model ofinduced-neuropathy model.

The diabetic animals have been treated by different doses of IL-6 (1, 3,10, and 30 μg/kg) on 30 days chronically. The treatment has beenadministrated in sub-cutaneous every day (study A), 3 times a week(study B) and once a week (study C) starting 10 days after the inductionuntil the sacrifice of the animal 40 days after the STZ-induction. Thesetreatments could be considered as a curative treatment, as IL-6 has beenadministered after the first molecular damages caused by a prolongedhyperglycemia.

The present protocol shows that a IL-6 treatment of 30 days whatever theschedule of administration, induces a neuroprotection against thediabetic neuropathy. The behavioral analyses with tail flick and the EMGtesting (sensory and motor velocities) show the neuroprotective effectof IL-6 administered by subcuteanous route.

The neuroprotective effect is focused on the sensory fibers as well asmotor fibers (the CMAP velocity was not altered when the animals weretreated with IL-6 compound). The compound prevents fibers from loss ofthe myelin sheath and degeneration.

In comparison with a daily IP treatment (see previous study performed atNeurofit, August 2001), IL-6 administered daily by subcutaneous routeseems to be as efficient as intraperitoneal treatment (see example 2) atall tested doses. Furthermore, a lower dosage characterized by a IL-6administration one or three times per week, do not lead to a decrease ofthe neuroprotective effect of the compound. It seems that the treatmentat 3 times per week displays the best neuroprotective effect (espaciallyat 10 and 30 μg/kg) without any side effect on the general behavioral ofthe STZ animals.

REFERENCES

-   1. Altschul S F et al, J Mol Biol, 215, 403-410, 1990-   2. Altschul S F et al, Nucleic Acids Res., 25:389-3402, 1997-   3. Britland S T, Young R T, Sharma A K, and Clarke B F. Diabetes,    39, 898-908. (1990)-   4. Breighton, B and Hayden, MR: S Afr Med J. 1981 Feb. 21; 59(8):    250.-   5. Chebath, J., Fischer, D., Kumar, A., Oh, J. W., Kollet, O.,    Lapidot, T., Fischer, M., Rose-John, S., Nagler, A., Slavin, S. and    Revel, M. Eur. Cytokine Netw. 1997 8,359-365.-   6. Devereux J et al, Nucleic Acids Res, 12, 387-395, 1984.-   7. Emerich D F, Cain C K, Greco C, Saydoff J A, Hu Z Y, Liu H,    Lindner M D Cell Transplant. 1997 May-June;6(3):249-66.-   8. Emerich, D. F., Lindner, M. D., Winn, S. R., Chen, E.-Y.,    Frydel, B. R., and Kordower, J. H. (1996). J. Neurosci., 16,    5168-5181.-   9. Emerich D F, Winn S R, Hantraye P M, Peschanski M, Chen E Y, Chu    Y, McDermott P, Baetge E E, Kordower J H Nature. 1997 Mar.    27;386(6623):395-9.-   10. Emerich D F, Hammang J P, Baetge E E, Winn S R Exp Neurol. 1994    November; 130(1):141-50.-   11. Fischer M, Goldschmitt J, Peschel C, Brakenhoff J P, Kallen K J,    Wollmer A, Grotzinger J, Rose-John S. Nat Biotechnol. 1997 February;    15(2):142-5-   12. Fisher et al., J. Neuroimmunology 119 (2001) 1-9-   13. Frei et al., J. Neuroimmunol., 31:147 (1991)-   14. Halimi H, Eisenstein M, Oh J, Revel M and Chebath J. Eur.    Cytokine Netw. 1995, 6: 135-143,-   15. Hirano et al, 1986 Nature (London) 234-73 (1986)-   16. Hirano T, Matsuda T and Nakajima K: Stem cells 1994, 12:262-277.-   17. Hirota H, Kiyama H, Kishimoto T. Taga T J Exp Med. 1996 Jun.    1;183(6):2627-34.-   18. Ishikawa et al., 1999, Cell Mol Neurobiol. 19, 587-96-   19. Lin B, Nasir J, Kalchman M A, McDonald H, Zeisler J, Goldberg Y    P, Hayden M R Genomics. 1995 Feb. 10;25(3):707-15.-   20. May et al, Proc Natl Acad Sci USA 83:8957 (1986);-   21. Mendel, I., Katz, A., Kozak, N., Ben-Nun, A. and Revel, M.    Eur. J. Immunol. 1998 28, 1727-1737.-   22. Murakami M, Hibi M, Nakagawa N, Nakagawa T, Yasukawa K,    Yamanishi K, Taga T, Kishimoto T Science. 1993 Jun.    18;260(5115):1808-10.-   23. Novick, D., Shulman, L. M., Chen, L. and Revel, M. Cytokine 1992    4, 6-11.-   24. Novick D, Shulman L M, Chen L and Revel M. Cytokine 1992, 4:    6-11.-   25. Novick D. Engelmann H. Wallach D. Leitner O. Revel M.    Rubinstein M. Journal of Chromatography 1990. 510:331-7.-   26. Paonessa G, Graziani R, Deserio A, Savino R, Ciapponi L, Lahmm    A, Salvati A L,-   27. Toniatti C and Ciliberto G. EMBO J. 1995: 14: 1942-1951.-   28. Pearson W R, Methods in Enzymology, 183, 63-99, 1990-   29. Pearson W R and Lipman D J, Proc Nat Acad Sci USA, 85,    2444-2448,1988-   30. Rakieten, N., Rakieten, M. L., and Nadkami, M. V., Studies on    the diabetogenic action of streptozotocin, Cancer Chemother. Rep.,    1963, 29, 91.-   31. Rudas B. Streptozotocin. Azmeimittel-Forschung, 22, 830-861.    (1972)-   32. Smith and Waterman J Mol Biol, 147,195-197, 1981, Advances in    Applied Mathematics, 2, 482489, 1981.-   33. Taga, T., Hibin M., Hirata, Y., Yamasaki, K., Yasukawa, K.,    Matsuda, T., Hirano, T. and Kishimoto, T. Cell 1989 58, 573-581.-   34. Toulmond, S., Vige, X., Fage, D., and Benavides, J. Neurosci    Lett 1992, 144, 49-52.-   35. Ward L D, Howlett G J, Discolo G, Yasukawa K, Hammacher A,    Moritz R L and Simpson R J. High affinity interleukin-6 receptor is    a hexameric complex consisting of two molecules each of    interleukin-6, interleukin-6 receptor and gp130. J. Biol. Chem.    1994, 269: 23286-23289.-   36. Yamada, M., and Hatanaka, H.: Brain Res 1994, 643, 173-80.-   37. Zilberstein et al, EMBO J. 5:2529 (1986)

1. (Cancelled
 2. A method according to claim 20, wherein the diabeticneuropathy is a polyneuropathy.
 3. A method according to claim 20,wherein the diabetic neuropathy is a mononeuropathy.
 4. A methodaccording to claim 20, wherein said substance is: a) IL-6; b) a fragmentof a) which binds to gp80 and initiates signaling through gp130; c) avariant of a) or b) which has at least 70% sequence identity with a) orb) and which initiates signaling through gp130; d) a variant of a) or b)which is encoded by a DNA sequence which hybridizes to the complement ofthe native DNA sequence encoding a) or b) under moderately stringentconditions and which initiates signaling through gp130; or e) a salt,fused protein or functional derivative of a), b), c) or d) whichinitiates signaling through gp130.
 5. A method according to claim 4,wherein said IL-6 is recombinant IL-6.
 6. A method according to claim20, wherein said substance is a) an IL-6R/IL-6 chimera-; b) a fragmentof a) which binds to and initiates signaling through gp130; c) a variantof a) or b) which has at least 70% sequence identity with a) or b) andwhich initiates signaling through gp130; d) a variant of a) or b) whichis encoded by a DNA sequence which hybridizes to the complement of theDNA sequence encoding a) or b) under moderately stringent conditions andwhich initiates signaling through gp130; or e) salt, fused protein orfunctional derivative of a), b), c) or d) which initiates signalingthrough gp130.
 7. A method according to claim 20, wherein the substanceis glycosylated at one or more sites.
 8. A method according to claim 20,wherein the substance is not glycosylated.
 9. A method according toclaim 4, wherein the fused protein comprises an immunoglobulin (Ig)fusion.
 10. A method according to claim 4, wherein the functionalderivative comprises at least one moiety attached to one or morefunctional groups which occur as one or more side chains on the aminoacid residues.
 11. A method according to claim 10, wherein the moiety isa polyethylene moiety.
 12. A method according to claim 20, wherein thesubstance signaling through gp130 is used in an amount ranging fromabout 0.1 to 1000 μg/kg.
 13. A method according to claim 12, wherein thesubstance signaling through gp130 is used in an amount of about 1 μg/kgor 3 μg/kg or 10 μg/kg or 30 μg/kg.
 14. A method according to claim 20,wherein the substance signaling through gp130 is administered daily. 15.A method according to claim 20, wherein the substance signaling throughgp130 is administered three times per week.
 16. A method according toclaim 20, wherein the substance signaling through gp130 is administeredonce a week.
 17. A method for the treatment and/or prevention ofdiabetic neuropathy, comprising administering a vector for inducingand/or enhancing the endogenous production of IL-6 in a cell.
 18. Amethod for the treatment and/or prevention of diabetic neuropathy,comprising administering a cell that has been genetically modified toproduce a substance signaling through gp130.
 19. A method for thetreatment and/or prevention of diabetic neuropathy, comprisingadministering an expression vector comprising the coding sequence of asubstance signaling through gp130.
 20. A method for treating and/orpreventing diabetic neuropathy, comprising administering to a patient inneed thereof an effective amount of a substance signaling through gp130.21. A method according to claim 4, wherein the diabetic neuropathy is apolyneuropathy.
 22. A method according to claim 4, wherein the diabeticneuropathy is a mononeuropathy.
 23. A method according to claim 4,wherein the substance is glycosylated at one or more sites.
 24. A methodaccording to claim 4, wherein the substance is not glycosylated.
 25. Amethod according to claim 4, wherein the fused protein comprises an IL-6receptor fusion.
 26. A method according to claim 4, wherein thesubstance signaling through gp130 is used in an amount ranging fromabout 0.1 to 1000 μg/kg or about 1 to 500 μg/kg or less than about 100μg/kg.
 27. A method according to claim 26, wherein the substancesignaling through gp130 is used in an amount of about 1 μg/kg or 3 μg/kgor 10 μg/kg or 30 μg/kg.
 28. A method according to claim 4, wherein thesubstance signaling through gp130 is administered daily.
 29. A methodaccording to claim 4, wherein the substance signaling through gp130 isadministered three times per week.
 30. A method according to claim 4,wherein the substance signaling through gp130 is administered once aweek.
 31. A method according to claim 20, wherein the substancesignaling through gp130 is used in an amount ranging from about 1 to 500μg/kg.
 32. A method according to claim 20, wherein the substancesignaling through gp130 is used in an amount ranging from about 0.1 to100 μg/kg.
 33. A pharmaceutical composition comprising apharmaceutically acceptable carrier, diluent or excipient and, as activeingredient, a unit dose of IL-6 corresponding to a dose selected fromthe group consisting of 1, 3, 10 and 30 μg/kg.
 34. A pharmaceuticalcomposition according to claim 33, wherein said unit dose of IL-6corresponds to a dose of 1 μg/kg.
 35. A pharmaceutical compositionaccording to claim 33, wherein said unit dose of IL-6 corresponds to adose of 10 μg/kg.
 36. A pharmaceutical composition according to claim33, wherein said unit dose of IL-6 corresponds to a dose of 10 μg/kg.37. A pharmaceutical composition according to claim 33, wherein saidunit dose of IL-6 corresponds to a dose of 35 μg/kg.